Glycomacropeptide medical foods for nutritional management of phenylketonuria and other metabolic disorders

ABSTRACT

Medical foods containing glycomacroprotein and additional supplemented amounts of arginine, leucine, and optionally other amino acids, such as tyrosine, are disclosed. The medical foods can be used to provide the complete protein requirements for patients having metabolic disorders such as phenylketonuria.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.13/795,125 filed Mar. 12, 2013, which is a continuation of Ser. No.12/813,988 filed on Jun. 11, 2010 and has patented as U.S. Pat. No.8,604,168, which claims the benefit of U.S. Provisional Application No.61/186,690 filed on Jun. 12, 2009. Each of these applications isincorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under DK071534 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

This invention relates generally to medical foods used for thenutritional management of metabolic disorders such as phenylketonuria.In particular, the present invention is directed to medical foodscontaining glycomacropeptide as a primary protein source supplementedwith additional amounts of the amino acids arginine, leucine, andtyrosine.

BACKGROUND OF THE INVENTION

Phenylalanine (Phe) is an indispensable amino acid that is converted totyrosine by the enzyme phenylalanine hydroxylase (PAH; EC 1.14.16.1) inan individual with normal metabolism. About 1 in every 15,000 infantsborn annually have absent or impaired function of this enzyme and arediagnosed with the metabolic disorder phenylketonuria (PKU) (Scriver C.R. 2001, The Metabolic & Molecular Bases of Inherited disease, 8th ed.New York: McGraw-Hill). If the diet of an individual with PKU is notmodified within the first 20 days of life, Phe and its breakdownproducts accumulate in the blood and brain, causing neurological damageand cognitive disability.

Dietary management of PKU requires a low-Phe diet, suggested for life.Foods such as meat, dairy, legumes, and bread must be avoided byindividuals with PKU because of the high Phe content. Although somelow-protein natural foods are allowed in the low-Phe diet (mainlycertain fruits and vegetables), the majority of dietary protein in thestandard PKU diet is typically supplied by a Phe-free amino acidformula. A daily tally of total Phe consumption for adults older than 19years of age must not exceed a target value from 220 to 770 mg/day forfemales or 290 to 1,200 mg/day for males (Acosta P & Yanicelli S. 2001,Protocol 1-Phenylketonuria (PKU), in Division, R. P., (editor), The RossMetabolic Formula System Nutrition Support Protocols. 4th ed.: RossProduct Division).

The standard amino acid formula-based diet for PKU is difficult tofollow, restrictive, and unpalatable. Non-compliance, a common problemwith the standard PKU diet, can cause severe neuropsychologicalimpairment. Thus, there is a need in the art for medical foods that aremore palatable than the standard amino-acid based formulas and thatprovide PKU patients with necessary protein, including essential aminoacids, while effectively maintaining low Phe levels in the blood andbrain.

SUMMARY OF THE INVENTION

The present invention provides medical foods designed to increasedietary compliance and quality of life for individuals with metabolicdisorders such as PKU. In one aspect, the invention encompasses medicalfoods for the management of a metabolic disorder. Such foods containglycomacropeptide (GMP) and additional supplemented amounts of certainamino acids, including arginine and leucine. Preferably, the weightratio within the medical foods of the amino acid arginine to the totalprotein is from about 60 to 90 milligrams arginine/gram total protein;more preferably, the weight ratio is about 75 milligrams arginine/gramtotal protein. Preferably, the weight ratio within the medical foods ofthe amino acid leucine to the total protein is from about 100 to 200milligrams leucine/gram total protein; more preferably, about 100milligrams leucine/gram total protein.

In certain embodiments, the medical foods additionally contain asupplemented amount of the amino acid tyrosine. Preferably, the weightratio within the medical foods of the amino acid tyrosine to the totalprotein is from about 62 to 93 milligrams tyrosine/gram total protein;more preferably, about 85 milligrams tyrosine/gram total protein.

In some embodiments, the total weight of the additional supplementedamino acids within the medical foods is from about 22% to 38% of thetotal weight of the protein from GMP and the supplemented amino acidstogether. The foods may be additionally supplemented with other aminoacids, including without limitation histidine and tryptophan.Preferably, in embodiments further containing supplemented amounts ofthe amino acids tryptophan and histidine, the total weight of theadditional supplemental amino acids is from about 25% to 42% of thetotal weight of the protein from the GMP and the supplemented aminoacids together. Optionally, the medical food is not additionallysupplemented with the amino acid methionine.

In certain embodiments, the medical foods encompassed by the inventionmay be targeted to specific metabolic disorders by varying the preferredcombination of additional supplemental amino acids contained in thefoods. For example, for the management of a phenylalanine metabolismdisorders such as phenylketonuria, the foods preferably containsupplemental amounts of the amino acids arginine, leucine, and tyrosinein addition to the GMP. However, for the management of tyrosinemetabolism disorders such as tyrosinemia, the foods would not containany supplemental amount of tyrosine.

The medical foods encompassed by the invention may be in the form of avariety of standard food products. Preferred forms include beverages,bars, wafers, puddings, gelatins, crackers, fruit leathers, nut butters,sauces, salad dressings, crisp cereal pieces, flakes, puffs, a pellets,or extruded solids.

In certain embodiments, the medical foods may be heat-treated duringproduction, such as, for example, by baking the foods. The inventorshave determined that during heat treatment, amino acid levels maydecrease; particularly the amino acid levels of any added tryptophan,tyrosine, histidine, leucine, or arginine. Accordingly, in some suchembodiments, the amount of initial additional supplemental amino acidsused to make the medical foods is higher than for foods that are notheat-treated, so that the final weight ratio falls within the preferredranges. As non-limiting examples, in preferred embodiments, the initialweight ratio of the amino acid tryptophan to total protein within themedical food before heat treatment is greater than about 12 milligramstryptophan/gram total protein, the initial weight ratio of the aminoacid tyrosine to total protein within the medical food before heattreatment is greater than about 85 milligrams tyrosine/gram totalprotein, the initial weight ratio of the amino acid histidine to totalprotein within the medical food before heat treatment may be greaterthan about 23 milligrams histidine/gram total protein, the initialweight ratio of the amino acid leucine to total protein within themedical food before heat treatment is greater than about 100 milligramsleucine/gram total protein, and/or the initial weight ratio of the aminoacid arginine to total protein within the medical food before heattreatment is greater than about 75 milligrams arginine/gram totalprotein.

In such exemplary embodiments, the initial amino acid levels within themedical foods may be such that the final weight ratio of the amino acidtryptophan to total protein within the medical food after heat treatmentis from about 12 to 14 milligrams tryptophan/gram total protein, thefinal weight ratio of the amino acid tyrosine to total protein withinthe medical food after heat treatment is from about 62 to 93 milligramstyrosine/gram total protein, the final weight ratio of the amino acidhistidine to total protein within the medical food after heat treatmentis from about 20 to 24 milligrams histidine/gram total protein, thefinal weight ratio of the amino acid leucine to total protein within themedical food after heat treatment is from about 100 to 200 milligramsleucine/gram total protein, and/or the final weight ratio of the aminoacid arginine to total protein within the medical food after heattreatment is from about 60 to 90 milligrams arginine/gram total protein.

The medical foods of the invention that are used in a PKU managementdiet must contain very low phenylalanine levels. Accordingly, in certainpreferred embodiments, the GMP included in the medical foods containsnot more than 2.0 milligrams phenylalanine contaminant per gram GMPprotein. Although GMP of such purity may be provided by a commercialvendor, in certain such embodiments, the GMP may be purified before itis included in the medical food. Because of the addition of supplementalamounts of amino acids to the foods that are not present in purifiedGMP, in certain preferred embodiments, the medical food of theinventions contains less than 1.5 milligrams phenylalanine per gramtotal protein. Certain non-protein ingredients, such as chocolate, maycontribute trace amounts of phenylalanine to the medical foods;accordingly, in certain embodiments, the medical foods contain fromabout 1.2 to about 1.8 milligrams phenylalanine per gram total protein.

The medical foods of the invention that are used in managing a tyrosinemetabolism disorder must contain very low phenylalanine plus tyrosinelevels. Accordingly, such embodiments do not contain supplementedamounts of tyrosine. Preferably, in embodiments for managing a tyrosinemetabolism disorder, the medical food contains less than 2.0 milligramsphenylalanine and tyrosine together per gram total protein.

In some embodiments for managing a tyrosine metabolism disordercontaining supplemental amounts of the amino acids arginine and leucine,the total weight of the additional supplemented amino acids within themedical foods is from about 16% to 29% of the total weight of theprotein from GMP and the supplemented amino acids together. In yet othersuch embodiments for managing a tyrosine metabolism disorder, inaddition to leucine and arginine, the medical foods contain additionalsupplemented amounts of the amino acids histidine and tryptophan.Preferably, in such embodiments, the total weight of the additionalsupplemental amino acids is from about 19% to 33% of the total weight ofthe protein from the GMP and the supplemented amino acids together. Tomaintain recommend supplementation levels of the other supplementedamino acids in the absence of tyrosine, additional GMP may be added tothe medical foods.

In a second aspect, the invention encompasses methods of making themedical foods described previously. The method includes the steps ofproviding glycomacropeptide (GMP) and additional supplemented amounts ofcertain amino acids, including arginine and leucine, and mixing theprovided materials with one or more non-protein ingredients to make afood. Preferably, the weight ratio of the amino acid arginine providedto the total protein provided is from about 60 to 90 milligramsarginine/gram total protein; more preferably, about 75 milligramsarginine/gram total protein. Preferably, the weight ratio of the aminoacid leucine provided to the total protein provided is from about 100 to200 milligrams leucine/gram total protein; more preferably, the ratio isabout 100 milligrams leucine/gram total protein.

In some preferred embodiments of the method, a supplemented amount ofthe amino acid tyrosine is also provided. Preferably, the weight ratioof the amino acid tyrosine provided to the total protein provided isfrom about 62 to 93 milligrams tyrosine/gram total protein; morepreferably, the ratio is about 85 milligrams tyrosine/gram totalprotein. In some such embodiments, the total weight of the additionalsupplemented amino acids is from about 22% to 38% of the total weight ofprotein from GMP and the supplemented amino acids together.

In certain embodiments, the method encompassed by the invention may bemodified to make medical foods targeted to specific metabolic disordersby varying the provided combination of supplemented amino acids. Forexample, for making medical foods used for the management of aphenylalanine metabolism disorders such as phenylketonuria, supplementalamounts of the amino acids arginine, leucine, and tyrosine in additionto the GMP are provided. However, for the making of medical foods usedfor the management of tyrosine metabolism disorders such as tyrosinemia,no supplemental amounts of tyrosine would be provided.

In certain preferred embodiments, the method includes the step ofpurifying the GMP so that it contains no more than 2.0 mg phenylalaninecontaminant per gram GMP protein. In some such embodiments, the step ofpurifying the GMP may be performed by one or more of the followingtechniques: cation exchange chromatography, ultrafiltration, anddiafiltration. Such embodiments may also include an additional step ofdrying the purified GMP by lyophilization or spray drying.

Certain embodiments may include the additional step of allowing the foodto set to form a pudding, gelatin, or fruit leather. Other embodimentsmay include the step of forming the food into a bar, a cracker, a flake,a puff, or a pellet, or extruding the food as an extruded solid.

Certain embodiments may include the additional step of heat-treating theprovided mixture when making the food. A non-limiting example of such astep is baking the food in an oven or other heated chamber. Theinventors have determined that certain amino acids, including tyrosine,tryptophan, arginine, leucine, and histidine, are lost or degradedduring heat treatment. Accordingly, in such embodiments, it is preferredthat the initial amounts of additional supplemental amino acids used tomake the medical foods are provided at higher levels than for foods thatare not heat-treated, so that as amino acids are lost or degradedthrough heat treatment, the final weight ratio falls within preferredranges.

As a non-limiting example, in preferred embodiments, the initial weightratio of the amino acid tryptophan provided to total protein providedbefore heat treatment may be greater than about 12 milligramstryptophan/gram total protein; the initial weight ratio of the aminoacid tyrosine provided to the total protein provided before heattreatment may be greater than about 85 milligrams tyrosine/gram totalprotein; the initial weight ratio of the amino acid histidine providedto total protein provided within the medical food before heat treatmentmay be greater than about 23 milligrams histidine/gram total protein;the initial weight ratio of the amino acid leucine provided to totalprotein provided within the medical food before heat treatment may begreater than about 100 milligrams leucine/gram total protein; and/or theinitial weight ratio of the amino acid arginine provided to totalprotein provided before heat treatment may be greater than about 75milligrams arginine/gram total protein.

During heat treatment, these amino acid levels within the food maydecrease. It is preferred that the final weight ratio of the amino acidtryptophan to total protein within the medical food after heat treatmentis from about 12 to 14 milligrams tryptophan/gram total protein, thefinal weight ratio of the amino acid tyrosine to total protein withinthe medical food after heat treatment is from about 62 to 93 milligramstyrosine/gram total protein, the final weight ratio of the amino acidhistidine to total protein within the medical food after heat treatmentis from about 20 to 24 milligrams histidine/gram total protein, thefinal weight ratio of the amino acid leucine to total protein within themedical food after heat treatment is from about 100 to 200 milligramsleucine/gram total protein, and/or the final weight ratio of the aminoacid arginine to total protein within the medical food after heattreatment is from about 60 to 90 milligrams arginine/gram total protein.

In a third aspect, the invention encompasses methods of treating ametabolic disorder, including without limitation a phenylalaninemetabolism disorder, a tyrosine metabolism disorder, a tryptophanmetabolism disorder, or a histidine metabolism disorder. These methodsinclude the step of administering to a human having a metabolic disordera medical food containing glycomacropeptide (GMP) and additionalsupplemented amounts of two or more amino acids, including arginine andleucine. Preferably, the weight ratio within the medical food of theamino acid arginine to the total protein is from about 60 to 90milligrams arginine/gram total protein; more preferably, the ratio isabout 75 milligrams arginine/gram total protein. Preferably, the weightratio within the medical food of the amino acid leucine to the proteinis from about 100 to 200 milligrams leucine/gram total protein; morepreferably, about 100 milligrams leucine/gram total protein.

In such embodiments where the medical food is not further supplementedwith tyrosine, the human treated with the medical food may have atyrosine metabolism disorder, including without limitation Type Ityrosinemia, Type II tyrosinemia, Type III tyrosinemia/Hawkinsinuria, orAlkaptonuria/Ochronosis. In such embodiments, the medical foodpreferably contains less than 2.0 milligrams phenylalanine and tyrosinetogether per grams total protein.

In yet other embodiments, the administered medical food additionallycontains a supplemented amount of the amino acid tyrosine. Preferably,the weight ratio within the medical food of the amino acid tyrosine tothe total protein is from about 62 to 93 milligrams tyrosine/gram totalprotein; more preferably, the ratio is about 85 milligrams tyrosine/gramtotal protein. In such embodiments, the human treated with the medicalfood may have a phenylalanine metabolism disorder, including withoutlimitation phenylketonuria (PKU), a tryptophan metabolism disorder,including without limitation hypertryptophanemia, or a histidinemetabolism disorder, including without limitation carnosinemia,histidinemia, or urocanic aciduria. In embodiments where a human with atryptophan metabolism disorder is treated, the administered medical fooddoes not contain supplemented amounts of tryptophan. In embodimentswhere a human with a histidine metabolism disorder is treated, theadministered medical food does not contain supplemented amounts ofhistidine.

In embodiments where the human being treated has a phenylalaninemetabolism disorder, the human is preferably at least two years old.Preferably, the medical food administered contains less than 1.5milligrams phenylalanine per gram total protein.

These and other features of the present invention will become apparentto the skilled artisan from the following detailed descriptionconsidered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows body weight as a function of time in weanling WT mice feddiets containing casein, GMP supplemented with limiting IAA (GMPadequate), or GMP supplemented with limiting IAA except Phe (GMP Phedeficient) over a 42 d. period. Values are means±SEM; n=10. Phe wasadded to the drinking water for the GMP Phe-deficient group (1 g Phe/L)on d 4 through the end of the study. There were no significantdifferences for changes in daily BW from d 14 to d 42.

FIG. 2 shows the concentration of Phe in 5 sections of brain,cerebellum, brain stem, hypothalamus, parietal cortex, and anteriorpiriform cortex, of PKU mice fed the GMP or amino acid (AA) diet for 47d. Values are means±SEM; n=8. *Different from AA, P≤0.001.

FIG. 3 shows cerebellum Phe levels for PKU mice fed the GMP or aminoacid (AA) diet for 47 d as a function of plasma threonine(Thr)+isoleucine (Iso)+valine (Val) levels.

FIG. 4 shows the amino acid profile of glycomacropeptide (BioPURE-GMP;Davisco Foods International Inc., LeSueur, Minn.) and casein (ALACID;New Zealand Milk Products, Santa Rosa, Calif.) expressed as g amino acidper 100 g product.

FIG. 5 shows mean Phe concentrations obtained after an overnight fastbefore breakfast from a single PKU subject fed an amino acid (AA) orglycomacropeptide (GMP) diet for 15 weeks at home. Data are shown for 6weeks of the 15-week study period when only foods with known Phe contentwere provided to the subject: weeks 3 and 15 (AA diet) and weeks 4, 7,11 and 13 (GMP diet). Phe concentrations in blood and plasma werecorrected for Phe intake and expressed as mmol Phe/L per 100 mg Pheintake. Phe concentration was determined using one of two methods, bloodspot collection analysed with tandem mass spectroscopy (MS/MS) andmeasurement of plasma Phe with an AA analyser. Values are means±SE; AAdiet (n=4 plasma Phe and n=4 blood Phe), GMP diet (n=4 plasma Phe andn=8 blood Phe). *Different from the AA diet, p<0.05.

FIG. 6 shows the concentration of total amino acids (AAs) and blood ureanitrogen in postprandial plasma with ingestion of the glycomacropeptide(GMP) or the AA diet. Plasma was obtained 2.5 h after eating breakfast;n=11 with the exception of blood urea nitrogen on study days 5 and 6 forwhich n=6. Total plasma AAs indicate the sum of all AAs measured inplasma. Values are means±SEMs. Total plasma AAs increased and blood ureanitrogen decreased with ingestion of the GMP diet when compared with day4 of the AA diet. There was a significant effect of time in therepeated-measures ANOVA. *Significantly different from the AA diet onday 4, P<0.05 (paired t test, pairing on subject).

FIG. 7 shows concentrations of phenylalanine in plasma of individualsubjects with phenylketonuria (n=11) after consuming the amino acid (AA)diet or the glycomacropeptide (GMP) diet for 4 d. Blood was obtained 2.5h after eating breakfast, and plasma was isolated for analysis of thecomplete AA profile. Subjects showed a range of plasma phenylalanineconcentrations after consuming the AA diet or the GMP diet for 4 d.There was no significant difference in the concentration ofphenylalanine in plasma when the last day of the AA diet (day 4) wascompared with the last day of the GMP diet (day 8); P=0.173 by paired ttest, pairing on subject. Group mean±SEM was 619±82 μmol/L (AA diet) and676±92 μmol/L (GMP diet). The mean change in the concentration ofphenylalanine in plasma was 57±52 μmol/L. phe, phenylalanine.

FIG. 8 shows the concentration of phenylalanine in postprandial (PP; 2.5h after eating breakfast) compared with fasting (fast, overnight fast)plasma in subjects with phenylketonuria fed glycomacropeptide (GMP)compared with the amino acid (AA) diet for 4 d. Group means and theresponse of individual subjects are shown; n=6 (day 4 compared with day8). There was no significant change in plasma phenylalanineconcentration comparing fasting with PP concentrations when consumingthe GMP diet (P=0.349); however, the AA diet showed a significantincrease in plasma phenylalanine (P=0.048) by paired t test, pairing onsubject. phe, phenylalanine.

FIG. 9 shows the concentrations of threonine and isoleucine inpostprandial plasma after consuming the glycomacropeptide (GMP) diet for4 d (days 5-8). Values are mean±SEM; n=11, of plasma obtained 2.5 hafter breakfast. For study days 3 and 4, all subjects consumed an aminoacid (AA) diet; on days 5-8, all AA formula was replaced with GMP foodproducts. There was a significant effect of time in therepeated-measures ANOVA. *Significantly different from the last day ofthe AA diet (day 4), P<0.05 (paired t test, pairing on subject).**Significantly different from the last day of the AA diet (day 4),P<0.0001. There was no further significant increase in plasmaconcentration of isoleucine and threonine after days 5 and 7,respectively. ile, isoleucine; thr, threonine.

FIG. 10 shows the amino acid profile of GMP strawberry pudding comparedto amino acid formula (Phlexy-10 Drink Mix, SHS North America,Rockville, Md., U.S.A.). Values are mean±SD. Sample size was n=2. Sameletter above GMP strawberry pudding and amino acid bars indicates thatvalues are not statistically different (P>0.05).

FIG. 11 is a bar graph showing acceptability ratings using fourdifferent criteria (odor, taste, after taste, and overall) for bothBettermilk™, a GMP food of the present invention, and Phenex-2™, acommonly used amino acid formula. The ratings are averaged from 27 nonPKU adults (unshaded bars) and 4 PKU adults (shaded bars). Values aremean±SD; * p<0.01, paired t-test. Acceptability ranking is: 1—dislikeextremely, 2—dislike a lot, 3—dislike, 4—dislike a little, 5—like alittle, 6-like, 7-like a lot, and 8-like extremely.

FIG. 12 is a bar graph showing plasma concentrations of ghrelin, insulinand amino acids for PKU subjects when on a GMP diet and on an amino aciddiet. Ghrelin and insulin values represent equal volumes of plasmacombined for each subject from days 3+4 for the AA breakfast, days 7+8for the GMP breakfast. Sum of postprandial (PP) plasma AA values on thelast day of the AA diet (day 4) and last day of the GMP diet (day 8).All values are means±SEM; n=6 for ghrelin fasting values. *Indicatessignificantly different from postprandial ghrelin with AA breakfast(p=0.03, paired t-test, pairing on subject; n=10). **Indicatesmoderately significant difference from insulin with the AA breakfast(p=0.053, paired t-test, pairing on subject; n=10). ***Indicatessignificantly different from sum of plasma AAs with the AA breakfast(p=0.049, paired t-test, pairing on subject; n=11).

FIG. 13 is a graph of the relationship between plasma ghrelinconcentrations 180 min after the start of breakfast (x axis) and feelingof fullness 2 h after breakfast (y axis) for PKU subjects on both GMP(unfilled circles) and amino acid (filled circles) diets. Lowerpostprandial ghrelin was associated with greater feeling of fullness.Lines represent least squares regression lines fitted to individual diettreatment data; AA breakfast is dashed line and GMP breakfast is solidline. Lines are significantly different. Using backward elimination witha mixed effects model, the best model predicting postprandial fullnessscores included diet treatment, postprandial ghrelin and the interactionbetween ghrelin and diet treatment.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby any later-filed nonprovisional applications.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

As used herein, the term “about” means within a range from 10% below to10% above a given value.

As used herein, the term “medical food” means “a food which isformulated to be consumed or administered enterally under thesupervision of a physician and which is intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, based on recognized scientific principles, areestablished by medical evaluation” (from section 5(b) of the Orphan DrugAct, 21 U.S.C. 360ee (b) (3)). Medical foods are distinguished from thebroader category of foods for special dietary use and from foods thatmake health claims by the requirement that medical foods be intended tomeet distinctive nutritional requirements of a disease or condition,used under medical supervision and intended for the specific dietarymanagement of a disease or condition.

The term “medical foods” does not pertain to all foods fed to sickpatients. Medical foods are foods that are specially formulated andprocessed (as opposed to a naturally occurring foodstuff used in anatural state) for the patient who is seriously ill or who requires theproduct as a major treatment modality. To be considered a medical food,a product must, at a minimum, meet the following criteria: the productmust be a food for oral or tube feeding; the product must be labeled forthe dietary management of a specific medical disorder, disease, orcondition for which there are distinctive nutritional requirements; andthe product must be intended to be used under medical supervision (fromU.S. Food and Drug Administration, Guidance for Industry: FrequentlyAsked Questions About Medical Foods, Center for Food Safety and AppliedNutrition, May 2007).

As used herein, a “supplemented amount” of an amino acid refers to thequantity of amino acid that is added to a mixture or contained in a foodthat does not come from (a) trace contamination of the GMP protein, or(b) trace amounts of amino acids contained in non-protein products. Anon-limiting example of a non-protein product is chocolate, which hastrace amounts of Phe, but is not recognized a s a significant source ofprotein or amino acids. Supplemented amounts may come from any othersource that is recognized as containing significant amounts of a givenamino acid or protein containing the amino acid, including withoutlimitation commercial amino acid supplements.

As used herein, “total protein” within a food means the aggregate of theprotein from the GMP within the food and the protein from additionalsupplemented amino acids within the food.

The following abbreviations are used throughout the present disclosure:AA, amino acid; Ala, alanine; Arg, arginine; Asn, asparagine; Asp,aspartic acid; BW, body weight; Cys, cysteine; DRI, dietary referenceintakes; Gln, glutamine; Glu, glutamic acid; Gly, glycine; GMP,glycomacropeptide; His, histidine; IAA, indispensible amino acid; Iso orIle, isoleucine; Leu, leucine; LNAA, large neutral amino acids; Met,methionine; MS/MS, tandem mass spectroscopy; PAH, phenylalaninehydroxylase; PE, protein equivalent; Phe, phenylalanine; PKU,phenylketonuria; Pro, proline; SEM, standard error of the mean; Ser,serine; Thr, threonine; Tyr, tyrosine; Trp, tryptophan; Val, valine; WT,wild type.

II. The Invention

The inventors have recently determined that medical foods made withglycomacropeptide protein supplemented with additional amounts of theamino acids arginine, histidine, leucine, and optionally, other aminoacids, as the amino acid/protein source contained in the foods, providea complete, low-Phe source of protein in the diet for individuals withPKU or other metabolic disorders. These foods are more palatable thanstandard AA formulas, and optimize the ability of GMP to lower levels ofPhe in the blood and brain. Accordingly, the present invention providesmedical foods, methods of making such foods, and methods ofadministering such foods as a protein source to individuals withmetabolic disorders such as PKU.

In one aspect, the invention provides medical foods containing acomplete low Phe protein source. The primary protein source in themedical foods of the present invention is Glycomacropeptide (GMP), anaturally occurring protein that contains no Phe in its pure form. GMPis formed during cheese making when chymosin specifically cleavesκ-casein between the 105 to 106 amino acid residues. Para-κ-casein(residues 1 to 105) coagulates, forming cheese curd, while GMP (residues106 to 169) remains in the whey. GMP is highly polar and is glycosylatedby galactosamine, galactose, and o-sialic acid at one or more threonineamino acid sites.

“GMP protein” refers to pure GMP polypeptide without the glycosylatingmoieties. GMP protein contains 47% (w/w) indispensable amino acids, butcontains no histidine (His), tryptophan (Trp), tyrosine (Tyr), arginine(Arg), Cysteine (Cys) or Phe.

A number of methods can be used to isolate GMP from whey. Detailedexamples of purification methods can be found, for example, in U.S. Pat.No. 5,968,586. Current large-scale technologies to isolate GMP from wheyuse ion exchange chromatography or ultrafiltration. GMP has anisoelectric point (pI) below 3.8, whereas other major whey proteins havepI values above 4.3. This physicochemical difference between GMP andother whey proteins is commonly used in isolation processes to separateGMP from whey.

Commercially available GMP contains Phe contaminants from residual wheyproteins. The amount of Phe contamination in commercial GMP varieswidely (i.e. 5 mg Phe/g product, manufacturer literature, Davisco FoodsIntl., Eden Prairie, Minn., U.S.A.; 2.0 mg Phe/g product, LacprodancGMP-20 manufacturer literature, Arla Foods, Arhus, Denmark).Traditional amino acid formula is free of Phe, which allows anindividual with PKU to consume natural foods that contain Phe to meettheir daily allowance. Preferred GMP for use in the present inventioncontains no more than 2.0 mg Phe/g GMP.

In certain preferred embodiments, commercially obtained GMP may bepurified to remove Phe contaminants before being used in the medicalfoods of the present invention. Possible purification processes are wellknown in the art, and include without limitation trapping contaminatingwhey proteins in crude GMP by adsorption onto a cation exchange resinand collecting the purified GMP in the flow-through fraction. Additionaltechniques known in the art, such as, for example,Ultrafiltration/diafiltration (UF/DF), can be used to concentrate theGMP and wash out peptides, salts, and nonprotein nitrogen. After thepurification and concentration steps, a number of techniques known inthe art can be used to dry the purified, concentrated GMP, includingwithout limitation lyophilization and spray drying.

Pure GMP contains no His, Tyr, Trp, Cys, Arg, or Phe, and is low inleucine (Leu). His, Trp, Phe, and Leu are all indispensable amino acids.Tyr, and Arg are conditionally indispensable amino acids, because Phe isa precursor to Tyr, and glutamate, proline and aspartate are precursorsto Arg. As a result, GMP as a primary protein source in foods must besupplemented to provide a nutritionally complete protein. The inventorshave determined optimal ranges for supplemental amounts of the aminoacids arginine, leucine, and tyrosine that are different than what hadbeen suggested previously in the art.

Accordingly, in certain preferred embodiments, the present inventionincludes medical foods for the management of a metabolic disorder wherethe medical foods contain glycomacropeptide (GMP) and additionaloptimized supplemental amounts of the amino acids arginine, leucineand/or tyrosine. Other amino acids may also be included in the medicalfoods of the invention. However, because the inventors have determinedthat methionine supplementation is not necessary, and in fact would makethe medical foods less palatable, in certain preferred embodiments, themedical foods of the invention do not contain an additional supplementedamount of the amino acid methionine. Amino acids approved for use infood products can be obtained from a variety of commercial sources knownin the art.

The preferred weight ratio within the medical food of each supplementedamino acids is expressed in the units of milligrams of that amino acidin the final medical food per gram of total protein, where a gram oftotal protein is defined as the sum of the protein from GMP (gnitrogen×6.25) and from the additional supplemented amino acids (gnitrogen×6.25). In certain preferred embodiments, the total weight ofthe additional supplemented amino acids is preferably from about 22% to38% of the total weight of the protein from GMP and the supplementedamino acids added together.

Preferably, the weight ratio within the medical foods of the amino acidarginine to the protein is from about 60 to 90 milligrams arginine/gramtotal protein; more preferably, the weight ratio within the medicalfoods of the amino acid arginine to the total protein is about 75milligrams arginine/gram total protein.

The preferred weight ratio within the medical foods of the amino acidleucine to the protein is from about 100 to 200 milligrams leucine/gramtotal protein; more preferably, the weight ratio within the medicalfoods of the amino acid leucine to the total protein is about 100milligrams leucine/gram total protein.

In those embodiments containing supplemental amounts of tyrosine, thepreferred weight ratio within the medical foods of the amino acidtyrosine to the total protein is from about 62 to 93 milligramstyrosine/gram total protein; more preferably, the weight ratio withinthe medical foods of the amino acid tyrosine to the total protein isabout 85 milligrams tyrosine/gram total protein.

In certain embodiments, the medical foods may optionally containadditional supplemented amino acids. For example, histidine and/ortryptophan may be included in the medical foods. For histidinesupplementation, the preferred weight ratio within the medical foods ofhistidine to the total protein is from about 20 to 24 milligramshistidine/gram total protein; more preferably, the weight ratio withinthe medical foods of the amino acid histidine to the total protein isabout 23 milligrams histidine/gram total protein.

For tryptophan supplementation, the preferred weight ratio within themedical foods of tryptophan to the total protein is from about 12 to 14milligrams tryptophan/gram total protein; more preferably, the weightratio within the medical foods of the amino acid tryptophan to the totalprotein is about 12 milligrams tryptophan/gram total protein.

In certain embodiments, the medical foods may be additionallysupplemented with essential vitamins and minerals, providing requirednon-protein nutritional supplementation in addition to a completeprotein source. Furthermore, the medical foods may contain a variety ofother low-Phe substances that are typically contained in conventionalfoods (non-protein ingredients).

The invention is not limited to medical foods for the treatment of Phemetabolism disorders such as PKU; instead the medical foods of theinvention additionally include foods for the management of a metabolismdisorder of other amino acids that are not present in GMP (i.e.metabolism disorders of His, Trp, Tyr, or Phe). For embodiments used inthe management of tyrosine metabolism disorders such tyrosinemia, theoptimal supplemental amounts of arginine and leucine are included in thefoods, but no supplemental amount of tyrosine is included. To adjust forthe lost tyrosine, the amount of GMP can be increased. In some suchembodiments, the total weight of the additional supplemented amino acidsis preferably from about 16% to 29% of the total weight of the proteinfrom GMP and the supplemented amino acids added together. Preferably,the amount of tyrosine plus phenylalanine together in such embodimentsis less than 2.0 mg per gram total protein.

The medical foods of the present invention encompass a wide variety offood types, including without limitation a formula, a beverage, a bar, awafer, a pudding, a gelatin, a cracker, a fruit leather, a nut butter, asauce, a salad dressing, a flake, a crisp cereal piece, a puff, apellet, or an extruded solid. These and other possible food types wouldbe easily recognized by those skilled in the art, and conventionalmanufacturing methods could be used to make the medical foods of theinvention using the ingredients of the invention along with otherlow-Phe substances typically used in conventional foods.

A number of the possible types of food encompassed by the invention aresubject to heat treatment during production. As a non-limiting example,crackers, bars, and crisp cereal pieces may be baked. Extruded solidsmay be heated prior to extrusion. Fruit leathers, sauces, and crispcereal pieces can be made by heating a mixture prior to cooling and, insome cases, drying the final product. Accordingly, in certainembodiments, the medical foods of the invention are heat-treated duringproduction.

The inventors have determined that heat treatment may lead to asignificant loss of the additional supplemented amino acids. Forexample, free amino acids such as Trp, Tyr, His, Leu, and Arg mayundergo the Maillard reaction. Light exposure can accelerate the Tyrphotodegradation reaction. Loss of the additional supplemented aminoacids by heat treatment or light exposure would increase the amount ofadditional supplemented amino acids that must be added to the medicalfoods. Thus, in some preferred embodiments, the initial weight ratio ofeach supplemented amino acid would be set higher for foods that are heattreated than for foods that are not heat-treated such that after lossthe final remaining amount of each supplemented amino acid falls withinthe preferred weight ratio.

In another aspect, the invention encompasses a method of making medicalfoods for the management of a metabolic disorders such as PKU. Themethod includes the steps of providing glycomacropeptide (GMP) andadditional supplemented amounts of amino acids including arginine andleucine, and mixing the provided materials with other substances to makethe foods. In certain embodiments of the method, the weight ratio of theamino acid arginine provided to the protein provided is from about 60 to90 milligrams leucine/gram total protein, preferably about 75 milligramsarginine/gram total protein. In certain embodiments of the method, theweight ratio of the amino acid leucine provided to the total proteinprovided is from about 100 to 200 milligrams leucine/gram total protein,preferably about 100 milligrams leucine/gram total protein.

As described above, a variety of other substances can be used in makingthe foods, including non-protein ingredients typically used to makeconventional foods. The other substances used, however, must be low-Pheor Phe-free substances.

In some embodiments, it is preferred that the total weight of theadditional supplemented amino acids used in the method is from about 22%to 38% of the total weight of the protein from GMP and the supplementedamino acids together. The method encompasses conventional techniquesused to make a variety of food types. As non-limiting examples, the foodmixture may be allowed to set to form a pudding, gelatin, or fruitleather; the food mixture may be formed into a bar, a cracker, a flake,a puff, or a pellet; or the food may be extruded as an extruded solid.In some embodiments of the method, the food mixture is heat treated.Examples of heat treatment include without limitation baking the foodmixture, pasteurizing the food mixture, boiling the heat mixture, orsubjecting the mixture to heated extrusion.

In certain embodiments of the method, the weight ratio of the amino acidtyrosine provided to the protein provided is from about 62 to 93milligrams tyrosine/gram total protein, preferably about 85 milligramstyrosine/gram total protein.

In certain embodiments of the method, the weight ratio of the amino acidhistidine provided to the protein provided is from about 20 to 24milligrams histidine/gram total protein, preferably about 23 milligramshistidine/gram total protein.

In certain embodiments of the method, the weight ratio within the foodof the amino acid tryptophan to the protein is from about 12 to 14milligrams tryptophan/gram total protein, preferably about 12 milligramstryptophan/gram total protein.

The method may also include the step of purifying the GMP so that itcontains not more than 2.0 mg phenylalanine contaminant per gram GMPprotein. A number of techniques known in the art can be used to purifythe GNP, including without limitation the use of cation exchangechromatography, ultrafiltration and diafiltration. The purified GNP mayfurther be dried using any one of a number of known drying techniques,including without limitation lyophilization or spray drying.

In yet another aspect, the invention encompasses a method of treating ametabolic disorder. This method includes the steps of selecting apatient with a metabolic disorder and administering to the patient amedical food comprising glycomacropeptide (GMP) and additional optimalsupplemented amounts of the amino acids arginine and leucine.Preferably, the weight ratio within the medical food of the amino acidarginine to the total protein is from about 60 to 90 milligramsarginine/gram total protein, more preferably about 75 milligramsarginine/gram total protein. Preferably, the weight ratio within thefood of the amino acid leucine to the total protein is from about 100 to200 milligrams leucine/gram total protein, more preferably about 100milligrams leucine/gram total protein.

The metabolic disorder is preferably one of a Phe metabolism disorder, aHis metabolism disorder, a Trp metabolism disorder, a Tyr metabolismdisorder, or a Phe metabolism disorder. In some embodiments, the totalweight of the additional supplemented amino acids in the administeredmedical food is from about 22% to 38% of the total weight of the GMPprotein and supplemented amino acids together.

In certain embodiments of the method, the medical food is notsupplemented with tyrosine, and the selected patient has a tyrosinemetabolism disorder. In such embodiments, the medical food preferablecontains less than 2.0 milligrams phenylalanine plus tyrosine per gramtotal protein. In some such embodiments, the total weight of theadditional supplemented amino acids in the administered medical food isfrom about 16% to 29% of the total weight of the GMP protein andsupplemented amino acids together.

In certain embodiments of the method, the medical foods used in themethod further contain additional optimal supplemented amounts of theamino acid tyrosine. Preferably, the weight ratio within the medicalfood of the amino acid tyrosine to the total protein is from about 62 to93 milligrams tyrosine/gram total protein, more preferably about 85milligrams tyrosine/gram total protein. If optimal amounts of arginine,leucine and tyrosine are included, the selected patient may have aphenylalanine metabolism disorder, a histidine metabolism disorder, or atryptophan metabolism disorder. If the patient has a histidinemetabolism disorder, the medical food administered does not contain asupplemented amount of histidine. If the patient has a tryptophanmetabolism disorder, the medical food administered does not contain asupplemented amount of tryptophan.

Subject to the limitations noted above, other amino acids may optionallybe included in the medical foods used in the method. In certainembodiments of the method, the weight ratio within the medical food ofthe amino acid histidine to the total protein is from about 20 to 24milligrams histidine/gram total protein, preferably about 23 milligramshistidine/gram total protein.

In certain embodiments of the method, the weight ratio within the foodof the amino acid tryptophan to the total protein is from about 12 to 14milligrams tryptophan/gram total protein, preferably about 12 milligramstryptophan/gram total protein.

In certain preferred embodiments, the patient selected has the metabolicdisorder PKU. The medical foods of the invention are more palatable thanconventional amino acid formulas, help decrease harmful Phe levels inthe plasma and brain, and help improve protein retention in suchpatients. In some embodiments, the food is administered to a human thatis at least two years old.

Although in certain preferred embodiments, the patient selected has themetabolic disorder PKU, the method encompasses the administration of themedical foods to patients having other metabolic disorders. Othermetabolic disorders that could be effectively treated by administeringthe foods of the present invention include: Tyrosine metabolismdisorders (Type I tyrosinemia, Type II tyrosinemia, Type IIItyrosinemia/Hawkinsinuria, and Alkaptonuria/Ochronosis); Tyrptophanmetabolism disorders (Hypertryptophanemia); and Histidine metabolismdisorders (Carnosinemia, Histidinemia, and Urocanic aciduria).

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

III. Examples Example 1: Supplemented Glycomacropeptide Diet in MurineModel of PKU

In this example, Applicants demonstrate that in a standard mouse modelof PKU, a supplemented glycomacropeptide diet supports growth andreduces phenyalanine concentrations in both plasma and brain, ascompared an amino acid diet. The murine model of PAH deficiency, thePah^(enu2) mouse (PKU mouse) is a suitable model to study thenutritional management of PKU as it exhibits hyperphenylalaninemia andcognitive defects similar to humans with PKU. Moreover, parallel to thehuman low-Phe diet in which the majority of dietary protein is providedby amino acids, studies in the PKU mouse utilize an amino acid-baseddiet often free of Phe with provision of Phe in drinking water. Ourobjective was to assess how ingestion of diets containing GMP as thesole protein source support growth and impact the concentrations ofamino acids, in particular Phe, in plasma and brain of wild-type (WT)and PKU mice. The results demonstated suitable growth and significantlyreduced concentrations of Phe in plasma and the brain of PKU mice fedGMP compared with an amino acid diet.

Materials and Methods.

Mice.

The animal facilities and protocols reported were approved by theUniversity of Wisconsin-Madison Institutional Animal Care and UseCommittee. Male and female 4- to 6-wk-old WT mice weighing 18-22 g werebred on the same background as the PKU mice (C57Bl/6, JacksonLaboratories). PKU mice were homozygous for the Pah mutation but werebred and backcrossed onto the C57Bl/6 background to increase breedingfacility. Breeding pairs of PKU mice were provided by Cary O. Harding,Oregon Health and Science University, Portland, Oreg. Genotyping for thepresence of the Pah^(enu2) mutation was performed by PCR analysis oftail biopsy DNA on an amplified region of exon 7. Mice were individuallyhoused in stainless steel, wire-bottom cages in a room maintained at 22°C. on a 12-:12-h light:dark cycle and were given free access to water.The mice were weighed every day at 1000 and food intake was determineddaily. At the conclusion of each experiment, mice were anesthetizedusing isoflurane via an anesthesia machine (IsoFlo, Abbott Laboratories)and killed by cardiac puncture/exsanguination between 0800 and 1000 withremoval of food 1 h before being killed.

Diets.

Purified diets were designed to provide similar amounts of vitamins,minerals, energy, and macronutrients (See Table 1). The protein sourcein the diets was provided by casein, free amino acids, GMP (BioPURE GMP,Davisco Foods), and GMP processed to reduce residual Phe content (seeEtzel M. R., J. Nutr. 2004; 134:S996-1002). The GMP diets weresupplemented with 1.5 times the NRC suggested requirement (see NRC,Nutrient Requirements of the Mouse, in Nutrient Requirements ofLaboratory Animals, 4th ed. Washington D.C.: National Academy Press;1995) for the following limiting IAA to compensate for faster absorptionand degradation of amino acids compared with intact protein: arginine,histidine, leucine, methionine, tryptophan, and tyrosine. The nitrogencontent of the amino acid and GMP low-Phe diets was similar, 24.1 and22.9 g nitrogen/kg diet, respectively, and both diets provided 175 gamino acids/kg of diet. Complete amino acid analysis of the diets wasconducted in the Experiment Station Chemical Laboratories, University ofMissouri-Columbia (Columbia, Mo.) (See Table 2).

TABLE 1 Experimental diets GMP GMP, Phe GMP, Amino acid, Casein adequatedeficient low Phe low Phe Ingredient g/kg Protein Casein 200.0 BioPureGMP¹ 200.0 200.0 Wisconsin GMP² 200.0 L-Arginine HCl 4.6 4.6 4.6 12.1³L-Cystine 3.0 3.5 L-Histidine, HCl—H₂0 3.2 3.2 3.2 4.5 L-Leucine 6.8 7.06.8 11.1 L-Methionine 7.0 7.0 7.0 8.2 L-Phenylalanine 10.4 0.15 0-2.5L-Tyrosine 5.0 5.0 5.0 5.0 L-Tryptophan 1.3 1.5 1.3 1.8 CarbohydrateSucrose 180.0 180.0 180.0 180.0 359.0 Cornstarch 302.0 279.0 287.0 288.0150.0 Maltodextrin 130.0 130.0 130.0 130.0 150.0 Cellulose 50.0 50.050.0 50.0 30.0 Fat Soybean oil 70.0 70.0 70.0 70.0 80.0 Cholinebitartrate 2.5 2.5 2.5 2.5 2.5 Vitamins and minerals Vitamin mixAIN-93-VX⁴ 10.0 10.0 10.0 10.0 13.0 Mineral mix AIN-93G-MX⁴ 35.0 35.035.0 35.0 35.0 Sodium chloride 8.0 Sodium phosphate dibasic 5.0 Calciumphosphate monobasic 5.0 5.0 5.0 8.0 Calcium carbonate 3.8 1.6 Magnesiumoxide 0.3 0.3 Antioxidant t-Butylhydroquinone 0.01 0.01 0.01 0.01 0.02¹BioPURE GMP; Davisco Foods International, Inc., LeSueur, MN.²Commercial GMP processed to reduce Phe content. ³In addition, thefollowing L-amino acids were included for a total of 175 g aminoacids/kg diet: alanine, 3.5; asparagine, 6.0; aspartic acid, 3.5;glutamic acid, 40; glycine, 23.3; isoleucine, 8.2; lysine HCl, 18.0;proline, 3.5; serine, 3.5; threonine, 8.2; and valine, 8. ⁴As reportedby Reeves et al, AIN-93 purified diets for laboratory rodents: finalreport of the American Institute of Nutrition ad hoc writing committeeon the reformulation of the AIN-76A rodent diet. J Nutr. 1993; 123:1939-51.

TABLE 2 Amino acid profile of diets GMP GMP, Phe GMP, Amino acid, Caseinadequate deficient low Phe low Phe Amino acid g amino acid/kg Alanine5.4 8.4 8.4 8.8 3.5 Arginine 6.8 1.2 3.8 3.7 8.4 Aspartic acid 12.6 13.012.9 14.3 9.7 Cysteine 3.4 0.2 0.2 0.2 3.5 Glutamic acid 41.2 32.3 31.933.5 40.0 Glycine 3.4 1.8 1.7 2.1 23.3 Histidine 5.7 2.6 2.9 2.9 2.6Isoleucine 9.3 16.0 15.5 15.1 9.0 Leucine 17.2 10.0 10.7 12.0 13.9Lysine 15.1 8.8 8.7 8.1 17.0 Methionine 4.8 8.3 7.9 8.3 8.2Phenylalanine 9.4 10.1 0.4 2.2 2.4 Proline 19.3 18.3 18.1 18.5 5.3Serine 8.0 8.9 9.2 10.4 2.8 Threonine 7.2 32.9 23.5 27.5 8.2 Tryptophan2.3 1.4 1.5 1.5 1.8 Tyrosine 8.6 4.2 4.0 4.9 4.1 Valine 12.0 12.5 12.314.1 9.4

Experimental Design.

Three experiments were conducted. Expt. 1 tested the adequacy of GMPsupplemented with IAA to support food intake and growth in 4-wk-old,male WT mice fed for 42 d. Three dietary treatment groups (n=10/group)were included: casein control, GMP supplemented with all limiting IAA(GMP adequate), and, to establish that Phe was limiting, GMP processedto reduce residual Phe and supplemented with all limiting IAA except Phe(GMP Phe deficient). When food intake failed after 3 d of feeding theGMP Phe-deficient diet, we added Phe to the drinking water (1 g Phe/L)on d 4.

Expt. 2 tested the ability of diets containing amino acids and GMP tosupport growth in male and female PKU mice (5-8 wk old) when Phe wasprovided in the drinking water (1 g Phe/L) for 21 d. Three dietarytreatment groups were included (n=10/group): PKU mice fed the GMPPhe-deficient diet, PKU mice fed an amino acid Phe-deficient diet, andWT mice fed the GMP-adequate diet. We measured drinking water intakedaily in PKU mice and adjusted for evaporation to determine the amountof Phe consumed.

Expt. 3 evaluated the ability of diets containing amino acids and GMPthat were supplemented with a minimum amount of Phe (determined fromExpt. 2) to support growth and affect the concentrations of amino acidsin plasma and the brain of male and female PKU mice fed for 47 d. Fourdietary treatment groups were included: 6-wk-old WT mice fed casein(n=8) or the GMP-adequate diet (n=7) and 8- to 10-wk-old PKU mice fedthe amino acid, low-Phe (n=10) or the GMP, low-Phe diet (n=11). Therewere similar numbers of male and female mice in each treatment group.Blood samples were obtained by orbital bleeding for amino acid analysisusing heparinized capillary tubes after 21 d of feeding (n=5/group).Mice were anesthetized, killed by cardiac exsanguination, anddecapitated after 47 d. The brains were quickly removed and placed on aglass plate cooled by dry ice. Using visual landmarks, samples weretaken from the following 5 regions; cerebellum, brain stem,hypothalamus, parietal cortex, and the anterior piriform cortex. Thesamples were placed in preweighed polystyrene tubes, weighed todetermine sample mass, and stored at −80° C. until processing.

Amino Acid Analysis.

Blood was collected by cardiac puncture into syringes containing a finalconcentration of 2.7 mmol/L EDTA and plasma was isolated bycentrifugation at 1700×g; 15 min at 4° C. The profile of free aminoacids in plasma was determined using a Beckmann 6300 amino acid analyzerequipped with an ion chromatography system using post column ninhydrinderivatization. The samples were deproteinized with sulfosalicylic acid,centrifuged (14,000×g; 5 min, and passed through a 0.2-μm syringe filterbefore adding an internal standard and injecting into the column.

The profile of free amino acids in the brain was determined in the AminoAcid Analysis Laboratory, University of California-Davis, School ofVeterinary Medicine (Davis, Calif.) using a Biochrom 30 amino acidanalyzer (Biochrom). The procedure for extraction of amino acids fromthe brain samples included the addition of 3% sulfosalicylic acidcontaining 100 μmol/L Norleucine as an internal standard (SigmaChemicals) in a ratio of 1:10 (wt:v), homogenization with an ultrasonicneedle for 2 min, centrifugation at 14,000×g; 20 min at 4° C., andfiltration of supernatant through a 0.45-μm syringe drive filter. Thefiltrate was adjusted to pH 2.2 with 0.4 mol/L LiOH and 0.05 mL wasinjected into the column. Values are expressed as nmol amino acid/g wettissue weight.

Statistics.

Statistical analyses were conducted using SAS version 8.2 (SASInstitute) and R (Universitat Wien, Vienna, Austria). Data were analyzedusing general linear models. The differences between dietary treatmentgroups were determined by the protected least significant differencetechnique. Statistics were performed on log-transformed data whenresidual plots indicated unequal variance among groups as occurred forsome of the data. Where appropriate, sex was included as a covariate toadjust for its potential influence. Changes in body weight (BW) amongtreatment groups were assessed with repeated measures analysis inExpt. 1. Among PKU mice, simple linear regression was used to examinethe correlations between dietary intakes of amino acids 48 h prior todeath and the concentrations of amino acids in plasma and brain. Allvalues are presented as means±SE; P≤0.05 was considered significant.

Results

Expt. 1.

Initial and final BW did not differ among the 3 dietary treatment groups(See FIG. 1). Food intake and BW did not differ when comparing thecasein and GMP adequate groups throughout the 42-d study. Mice stoppedeating the GMP Phe-deficient diet after 3 d, at which time Phe was addedto the drinking water and food intake resumed. The 3 dietary groups didnot differ in changes in daily BW from d 14 to d 42.

The profile of amino acids in plasma was significantly altered withintake of GMP compared with casein. WT mice fed the GMP-adequate orPhe-deficient diets showed elevated plasma concentrations of the IAA,threonine, isoleucine, and methionine, which were 3-fold, 2.4-fold, and1.6-fold, respectively, of the concentrations in mice fed the caseindiet (data not shown). Mice fed the GMP Phe-deficient diet showedsignificantly lower plasma concentrations of Phe and tyrosine comparedwith the GMP-adequate and casein groups.

Expt. 2.

Initial (16-18±1.4 g) and final (19-21±1.3 g) BW and food intake (3.3 to4.1±0.3 g/d) did not significantly differ among the 3 treatment groupsfor 21 d. Mean Phe intake in PKU mice was 6.5±0.5 mg Phe/d withingestion of the amino acid Phe-deficient diet and 5.9±0.3 mg Phe/d withingestion of the GMP Phe-deficient diet (P>0.10). Considering ourobservations from Expt. 1 and 2 that growth may be limited withprovision of Phe in the drinking water, we decided to supplement thelow-Phe amino acid and GMP diets for Expt. 3 to contain 2.5 g Phe/kgdiet. This provided a daily Phe intake for growing PKU mice of 7.5-10 mgPhe.

Expt. 3.

Gain in BW, feed utilization based on the ratio of feed intake to gainin BW, and the protein efficiency ratio did not differ among the 4dietary treatment groups (see Table 3). PKU mice were ˜2 g heavier thanWT mice (P<0.05), consistent with the former being 2 wk older. Femalemice of both genotypes weighed less than male mice at the end of thestudy (20±1 g vs. 25±1 g; n=17-18; P<0.0001).

TABLE 3 BW, feed utilization, and organ mass of WT and PKU mice feddiets containing casein, GMP, or amino acids (Expt. 3)¹ PKU mice WT miceGMP Amino acid, GMP, Casein adequate low Phe low Phe n 8 7 10 11 BWInitial, g 16.9 ± 0.8^(bc ) 16.3 ± 1.2^(c)  19.3 ± 1.0^(a )  18.6 ±0.9^(ab) Final, g 21.2 ± 1.1^(b)  21.2 ± 0.9^(b ) 23.8 ± 1.3^(a ) 23.3 ±0.8^(a ) Gain, g/47 d 4.3 ± 0.6  5.0 ± 0.7 4.5 ± 0.8 4.7 ± 0.7 Feedutilization Feed intake, g 147 ± 5^(b)  145 ± 4^(b )  169 ± 8^(a )  166± 4^(a )  Feed: gain ratio 39 ± 6   32 ± 3  45 ± 6  40 ± 6  Proteinefficiency ratio² 0.17 ± 0.02  0.20 ± 0.08 0.15 ± 0.02 0.16 ± 0.03Relative organ mass g/100 g BW Kidney 1.38 ± 0.04^(b)  1.28 ± 0.02^(bc) 1.48 ± 0.05^(a)  1.26 ± 0.02^(c) Heart 0.51 ± 0.01^(b)  0.60 ± 0.04^(a) 0.52 ± 0.01^(b)  0.52 ± 0.01^(b) Liver 4.61 ± 0.20^(b)  4.77 ± 0.13^(b) 5.22 ± 0.09^(a)  5.31 ± 0.11^(a) ¹Values are means ± SE. Means in a rowwith superscripts without a common letter differ, P < 0.05. ²Proteinefficiency ratio, g gain in BW/g protein intake.

Relative organ mass showed significant differences due to diet and sex.Kidney mass was significantly greater in PKU mice fed the amino aciddiet compared with the other groups. Heart mass was significantlygreater in WT mice fed the GMP-adequate diet compared with the othergroups. PKU mice fed either the amino acid or GMP diet showedsignificantly greater relative mass of liver compared with WT mice.Female mice of both genotypes showed significantly lower relative kidneymass and significantly greater relative heart mass compared with malemice.

The profile of amino acids in plasma was affected by diet and sex (seeTable 4).

TABLE 4 Concentrations of amino acids in plasma of WT and PKU mice feddiets containing casein, GMP, or amino acids (Expt. 3)¹ GMP PKU mice WTmice adequate Amino Acid GMP Amino Casein acid low Phe low Phe acidμmol/L Alanine 793 ± 97   570 ± 108 698 ± 63  580 ± 75  Arginine 86 ±7^(a) 61 ± 5^(b) 81 ± 7^(a) 62 ± 5^(b) Aspartate 30 ± 2  25 ± 3  23 ± 2 24 ± 2  Citrulline 46 ± 4  41 ± 3  57 ± 6  53 ± 6  Cystine  8 ± 2  8 ± 211 ± 3   8 ± 3 Glutamate 47 ± 4  44 ± 4  36 ± 4  41 ± 3  Glutamine  565± 41^(ab) 613 ± 35^(a) 430 ± 17^(c)  505 ± 25^(bc) Glycine 248 ± 15^(b)227 ± 21^(b) 654 ± 43^(a) 182 9^(b) Histidine 91 ± 8^(a) 65 ± 4^(b) 71 ±4^(b) 62 ± 4^(b) Isoleucine 95 ± 8^(b) 155 ± 31^(a) 78 ± 5^(b) 155 ±15^(a) Leucine 190 ± 11^(a) 123 ± 13^(b) 108 ± B^(b)  130 ± 8^(b) Lysine 501 ± 49^(a) 277 ± 12^(c) 397 ± 37^(b) 269 ± 16^(c) Methionine 96± 9   85 ± 13 80 ± 9  70 ± 9  Ornithine 63 ± 6^(a) 35 ± 2^(c) 47 ± 4^(b) 40 ± 3^(bc) Phenylalanine 59 ± 3^(c) 45 ± 3^(c) 851 ± 29^(a) 756 ±21^(b) Proline 209 ± 35^(a) 228 ± 83^(a) 74 ± 6^(b)  97 ± 11^(b) Serine 248 ± 24^(ab)  207 ± 23^(bc) 265 ± 14^(a) 173 ± 10^(c) Taurine 643 ±22  600 ± 67  624 ± 63  532 ± 45  Threonine 303 ± 32^(b) 562 ± 76⁸ 331 ±19^(b) 557 ± 65^(a) Tryptophan 110 ± 7^(a)   106 ± 16^(ab)  87 ± 10^(c) 93 ± 5^(bc) Tyrosine 121 ± 12^(a) 82 ± 7^(b) 31 ± 3^(c) 25 ± 2^(c)Valine 293 ± 31^(a) 228 ± 30¹ 184 ± 16^(c)  262 ± 22^(ab) BCAA² 609 ±49^(a) 505 ± 66^(a) 370 ± 16^(b) 548 ± 41^(a) ¹Values are means ± SE, n= 8. Means in a row with superscripts without a common letter differ, P< 0.05. ²BCAA, Sum of isoleucine, leucine, and valine.

PKU mice fed either the amino acid or GMP low-Phe diet showed 15-foldgreater plasma concentrations of Phe and a 60-70% decrease in plasmaconcentrations of tyrosine and proline compared with WT mice fed eitherthe casein or GMP diets. Both WT and PKU mice fed GMP diets showedplasma concentrations of threonine and isoleucine that were ˜2 times thevalues in WT and PKU mice fed the casein or amino acid diets (P<0.002).Decreased plasma concentrations of lysine were noted in WT and PKU micefed GMP diets (272±11 μmol/L) compared with WT and PKU mice fed thecasein or amino acid diets (443±31 μmol/L; P<0.0001; n=17-18). Femalemice of both genotypes showed greater plasma concentrations of tyrosine(74±6 vs. 53±8 μmol/L) and tryptophan (113±5 vs. 81±5 μmol/L) comparedwith male mice (P<0.01; n=17-18).

PKU mice fed GMP compared with the amino acid diet had significantdifferences in the concentrations of amino acids in plasma. PKU mice hada significant 11% decrease in the concentration of Phe in plasma withingestion of GMP compared with the amino acid diet for 47 d; this effectwas not observed at 21 d. Phe intake for the last 48 h before mice werekilled was similar for PKU mice (16-18 mg Phe/48 h) but wassignificantly lower compared with WT mice (58-66 mg Phe/48 h). The sumof plasma concentrations of the branched chain amino acids, isoleucine,leucine and valine, increased by 50% in PKU mice fed GMP compared withthe amino acid diet; however, the concentration of leucine did notdiffer. Among the PKU mice, dietary amino acid intake for the last 48 hbefore mice were killed and the concentrations of amino acids in plasmawere correlated. The highest positive correlations (P<0.0001; n=15)include the following: glycine, R²=0.88; threonine, R²=0.45; isoleucine,R²=0.44; and valine, R²=0.34.

The profile of amino acids in the cerebellum differed significantly dueto diet but not sex (see Table 5). The concentration of Phe incerebellum of PKU mice was 3 to 4 times the value in WT mice (P<0.0001).The concentrations of tyrosine and the sum of the branched chain aminoacids in cerebellum of PKU mice were −50% of that in WT mice regardlessof diet (P<0.0001). PKU mice fed the GMP diet had a 20% decrease in theconcentration of Phe in cerebellum compared with PKU mice fed the aminoacid diet.

Moreover, this response of a 20% decrease in Phe concentration was notedin each of 5 sections of brain sampled: cerebellum, brain stem,hypothalamus, parietal cortex, and anterior piriform cortex (FIG. 2).The concentrations of threonine and isoleucine in the cerebellumincreased 70-100% in PKU mice fed the GMP diet compared with the aminoacid diet (P<0.0001). A similar trend was noted for higher valineconcentration in the cerebellum of PKU mice fed the GMP diet comparedwith the amino acid diet (P<0.10). The concentration of Phe in thecerebellum of PKU mice was inversely correlated with the concentrationsof threonine, isoleucine, and valine in plasma as well as the sum of theconcentrations of threonine, isoleucine, and valine in plasma,R²=0.65-0.77 (P<0.0001) (FIG. 3). The concentration of glutamine incerebellum was 11% lower in PKU mice compared with WT mice regardless ofdiet (P<0.05). The concentrations in the cerebellum of tryptophan, theprecursor of the neurotransmitter serotonin, and glycine, a precursor ofthe glycinergic neurotransmitter system of the brain, did not differamong groups.

TABLE 5 Concentrations of amino acids in cerebellum of WT and PKU micefed diets containing casein, GMP, or amino acids (Expt. 3)¹ WT mice PKUmice GMP Amino acid GMP Casein adequate low Phe low Phe Amino acidnmol/g Alanine 2380 ± 193 2145 ± 134 2082 ± 27 2303 ± 83 Arginine 55 ± 850 ± 6 35 ± 3 40 ± 5 Aspartate 9060 ± 306 9122 ± 256 8406 ± 191 8836 ±630 Citrulline 373 ± 39 320 ± 42 293 ± 29 277 ± 25 Half-Cystine 110 ± 2299 ± 15 93 ± 21 149 ± 39 Glutamate 12,870 ± 837 13,264 ± 712 12,886 ±367 13,254 ± 505 Glutamine 11,459 ± 88^(a) 11,209 ± 197^(a) 10,049 ±359^(b) 10,099 ± 250^(b) Glycine 3923 ± 316 3764 ± 228 4383 ± 334 3753 ±291 Histidine 216 ± 17 186 ± 12 236 ± 9 200 ± 8 Isoleucine 197 ± 19^(a)192 ± 24^(a) 61 ± 21^(c) 123 ± 21^(b) Leucine 462 ± 66^(a) 366 ± 43^(a)164 ± 30^(b) 185 ± 30^(b) Lysine 683 ± 35^(a) 581 ± 38^(b) 678 ± 31^(a)615 ± 10^(ab) Methionine 309 ± 18 286 ± 24 214 ± 14 163 ± 24 Ornithine 5± 1 5 ± 2 3 ± 2 7 ± 4 Phenylalanine 271 ± 34^(c) 232 ± 22^(c) 1030 ±25^(a) 820 ± 49^(b) Proline 312 ± 29 255 ± 35 187 ± 15 218 ± 24 Serine1856 ± 91^(b) 1780 ± 87^(bc) 2156 ± 44^(a) 1609 ± 78^(c) Taurine 10,886± 58 11,109 ± 292 10,896 ± 108 10,603 ± 96 Threonine 1300 ± 49^(c) 2590± 87^(a) 1589 ± 46^(b) 2695 ± 82^(a) Tryptophan 90 ± 26 86 ± 12 59 ± 273 ± 3 Tyrosine 341 ± 27^(a) 278 ± 25^(b) 128 ± 14^(c) 128 ± 12^(c)Valine 337 ± 40^(a) 304 ± 27^(ab) 187 ± 11^(c) 246 ± 20^(bc) BCAA2 966 ±127^(a) 862 ± 92^(a) 413 ± 55^(b) 553 ± 39^(b) ¹Values are means ± SE, n= 8. For PKU mice, the sample size was 3 for the following amino acids:alanine, aspartate, citrulline, glutamine, proline, taurine, andtryptophan. Means in a row with superscripts without a common letterdiffer, P < 0.05. ²BCAA, Sum of isoleucine, leucine, and valine.

Discussion

This study assesses the ability of diets containing GMP supplementedwith IAA as the sole protein source to support growth and affect theconcentrations of amino acids in the plasma and brains of PKU mice. Insupport of utilization of GMP as a source of low-Phe protein in the PKUdiet, we observed similar growth with significantly lower concentrationsof Phe in the plasma and brains of PKU mice fed GMP compared with anamino acid diet.

When fed as the sole source of dietary protein, GMP contains limitingamounts of several IAA for growing mice including: arginine, histidine,leucine, methionine, Phe, tryptophan, and tyrosine. Our results showedadequate growth of mice that are fed GMP supplemented with theselimiting IAA. In Expt. 1, weanling WT mice fed casein or theGMP-adequate diet had virtually identical growth over 6 wk. In Expt. 3,PKU mice fed GMP or amino acid diets with similar Phe intake showedgains in BW, feed efficiency and a protein efficiency ratio that werenot significantly different. These data demonstrate that GMPsupplemented with limiting IAA provides a nutritionally adequate sourceof dietary protein for growing mice.

Consumption of a diet that is deficient in an IAA rapidly depresses theconcentration of the limiting IAA in plasma and brain with reduced foodintake in rats (Harper, et al., Physiol Rev. 1970; 50:428-39). Thus, itwas not surprising that in Expt. 1, mice stopped eating the GMPPhe-deficient diet and lost BW after only 3 d of this diet and thataddition of Phe to the drinking water normalized food intake and gain inBW. The plasma concentrations of isoleucine and threonine in WT mice fedthe GMP-adequate diet were 2 to 3 times those in WT mice fed the caseindiet. However, these alterations in plasma amino acid concentrations didnot impair food intake in mice fed GMP once the deficiency of Phe wascorrected. Thus, we conclude that ingestion of GMP supplemented with alllimiting IAA alters the plasma amino acid profile without reducing foodintake in growing mice.

In contrast to other IAA, hepatic uptake of threonine is low andoxidation of threonine to CO₂ via liver threonine dehydratase activity(EC 4.2.1.16) is limited in both humans (Darling et al., Am J PhysiolEndocrinol Metab. 2000; 278:E877-84) and rats (Harper, et al., PhysiolRev. 1970; 50:428-39). Thus, an increase in dietary threonine without anincrease in total protein intake results in expansion of the plasmathreonine pool without toxicity if diets provide<15 times the normallevel of threonine. The concentration of threonine in plasma showed thelargest increase with ingestion of GMP compared with the casein andamino acid diet in all 3 experiments.

Degradation of threonine to glycine via threonine dehydrogenase (EC1.1.1.103) is a major catabolic pathway in rats but not in humans(Darling, et al., Am J Physiol Endocrinol Metab. 2000; 278:E877-84).Elevated glycine levels are potentially neurotoxic in the brain due tothe glycinergic neurotransmitter system that can inhibit or stimulatetransmission of nervous impulses (Spencer et al., J. Neurosci. 1989;9:2718-36). However, our demonstration of no increase in theconcentration of glycine in both plasma and brain suggests that feedinga GMP diet that provides 3 times the normal intake of threonine is notsufficient to modify the concentration of glycine in brain. Takentogether, these findings support the safety of dietary GMP.

In addition, the 11% decrease in the concentration of Phe in plasma ofPKU mice fed GMP compared with the amino acid diet for 47 d is apositive finding for the nutritional management of PKU. The finding ofgreatest relevance to the management of PKU and the known neurotoxiceffects of Phe is our observation that PKU mice fed GMP compared withthe amino acid diet had a 20% decrease in the concentrations of Phe in 5sections of brain. The concentration of Phe in brain is the bestcorrelate of mental impairment in individuals with PKU (Donlon et al.,Metabolic and Molecular Basis of Inherited Disease, in Scriver et al.,editors, 8th ed. 77th chapter, Hyperphenylalaninemia: PhenylalanineHydroxylase Deficiency, New York: McGraw-Hill; 2007). The most likelyexplanation for the reduced concentration of Phe in the brain of PKUmice fed GMP is that elevated plasma levels of LNAA due to ingestion ofGMP competitively inhibit Phe transport across the blood brain barriervia the LNAA carrier protein that has a significantly lower Km in thebrain compared with the gut. This conclusion is supported by asignificant inverse correlation between higher plasma concentrations ofthreonine, isoleucine, and valine and lower brain concentration of Phe.Interestingly, previous research demonstrates that isoleucine, but notthreonine, competitively inhibits Phe transport in rat brain (Tovar etal., J. Neurochem. 1988; 51:1285-93).

In summary, we demonstrate that PKU mice fed a diet with 20% GMPsupplemented with IAA compared with an amino acid diet show similargrowth and lower concentrations of Phe in plasma and the brain. Thesedata establish that GMP can be formulated into a nutritionally adequatecomplete protein for growing mice and suggest that long-term feedingstudies may provide further insight into the metabolism of GMP. Ourfindings support continued research to establish the efficacy of foodsand beverages made with GMP in the nutritional management of PKU inhumans.

Example 2: Palatability of Foods Made with GMP and Supplemental AAs

In this Example, Applicants made a variety of palatable, low-phe foodsand beverages with GMP and assessed their acceptability by conductingconsumer sensory studies in individuals with PKU. Results demonstrateacceptability of products made with GMP.

Materials and Methods

Foods and Beverages.

Strawberry pudding, strawberry fruit leather, chocolate beverage, snackcracker and an orange sports beverage containing GMP were developed forthis study in the Food Applications Laboratory at the Wisconsin Centerfor Dairy Research (CDR), University of Wisconsin-Madison (UW). TheBioPURE-GMP (Davisco Foods International, Inc., LeSueur, Minn.) was usedto formulate GMP products. The amino acid profile of BioPURE-GMPcompared to casein is shown in FIG. 4.

A commercial amino acid-based chocolate beverage and low protein crackerwere included in the taste testing to provide comparisons between GMPproducts and products currently used in the PKU diet. The nitrogenconcentration of GMP and amino acid beverages was similar. The energyand protein content of all foods tested is provided in Table 6.

TABLE 6 Mean acceptability ratings of foods and beverages made with GMPas tested in individuals with PKU Product No. of subjects AppearanceOdor Taste Texture Overall A. Acceptability ratings for GMP pudding,fruit leather, and sports beverage GMP strawberry pudding 31 4.0 ± 0.73.6 ± 1.1 4.2 ± 1.0 4.1 ± 0.8 4.2 ± 0.9 GMP strawberry fruit leather 313.4 ± 0.8 3.8 ± 0.9 3.4 ± 1.1 3.0 ± 0.9 3.4 ± 1.0 GMP orange sportsbeverage 18 4.1 ± 0.6  36 ± 0.9 2.9 ± 1.2 3.8 ± 1.0 3.3 ± 1.1 B.Acceptability rating for GMP and amino acid-based chocolate beverage GMPchocolate beverage 32  3.8 ± 0.8^(a)  3.7 ± 1.0^(a)  3.2 ± 1.1^(a) 3.3 ±1.0  3.3 ± 1.0^(a) Amino acid chocolate beverage 32  3.1 ± 1.1^(b)  2.8± 1.3^(b)  2.2 ± 1.3^(b) 3.1 ± 1.1  2.5 ± 1.4^(b) C. Acceptabilityratings for GMP and a low-protein snack cracker GMP snack crackers 18 3.8 ± 0.6^(a)  3.9 ± 0.8^(a)  3.7 ± 0.8^(a) 3.6 ± 0.9 3.6 ± 1.4 Lowprotein crackers 18  3.2 ± 0.9^(b)  3.2 ± 0.8^(b)  2.9 ± 1.1^(b) 3.2 ±1.2 2.9 ± 1.3The energy and protein content of the foods tested is as follows: GMPstrawberry pudding, 213 kcal and 5.7 g protein per ½ cup serving (113g); GMP fruit leather, 60 kcal and 0.7 g protein per 15 g; GMP orangesports beverage, 67 kcal and 7.9 g protein per 8 oz (234 g); GMPchocolate beverage, 148 kcal and 10.2 g protein per 8 oz (236 g); aminoacid chocolate beverage, 187 kcal and 11.4 g protein per 8 oz (202 g);GMP snack crackers, 110 kcal and 1.3 g protein per 30 g; low proteincrackers, 135 kcal and 0.1 g protein per 30 g. Means±standard deviation(score reference: 1, dislike very much; 2, dislike; 3, neither like nordislike; 4, like; 5, like very much). Means with different lettersuperscripts (a or b) in the same column for B. or C. do significantlydiffer at p 0.05.

Sensory Studies to Assess Acceptability of Foods and Beverages.

The protocol for sensory studies was approved by the Social andBehavioral Sciences Institutional Review Board, UW. Three sensorystudies were performed with PKU subjects attending PKU Summer Camps in2004 and 2005 and a PKU family conference in 2005 (n=49; age range 12-42years). The studies were conducted in the Sensory Analysis Laboratory,Department of Food Science, UW or at the Waisman Center.

Test samples of 20-30 g were presented to subjects in balanced randomorder with three digit blind codes. Foods and beverages were rated usinga five-point hedonic scale (1=dislike very much, 2=dislike, 3=neitherlike nor dislike, 4=like, 5=like very much) to evaluate five sensorycategories, including appearance, odor, taste, texture and overallacceptability.

Statistical Analysis.

Dependent t-test was performed to analyze mean acceptability scores forGMP chocolate beverage and amino acid chocolate beverage. Group meanswere considered to be significantly different at p 6 0.05, as determinedby a two-tailed t-test using Statistical Analysis Software package (SASInstitutes Inc., Version 9.1.3, Cary, N.C., USA). Acceptability scoresfor GMP snack crackers and low protein crackers were compared usinggeneral linear model procedure (PROC GLM) followed by Fisher's leastsquare means for mean separation. Data are presented as means withstandard deviations.

Results

PKU subjects tasted a total of 7 products during PKU events in 2004 and2005 (Table 6). Among these foods and beverages, GMP strawberry puddingwas the most acceptable (overall score of 4.2±0.9) and other foods inorder of overall acceptability were GMP snack crackers (3.6±1.4), GMPstrawberry fruit leather (3.4±1.0), GMP chocolate beverage (3.3±1.0),GMP orange sports beverage (3.3±1.1), and low protein crackers(2.9±1.3). An amino acid chocolate beverage was least acceptable(2.5±1.4). A score of <3 indicates that a food or beverage isunacceptable with respect to a specific category, whereas a score of 3indicates neutral acceptance.

PKU subjects rated the appearance, odor, taste and overall acceptabilityof GMP chocolate beverage as significantly more acceptable compared tothe amino acid based beverage (p 0.05, Table 6B). Appearance, odor andtaste of the GMP snack crackers were rated as significantly moreacceptable compared to the low protein cracker (p≤0.05), but overallacceptability was not significantly different between the two types ofcrackers (Table 6C).

Discussion

These data demonstrate that the functional properties of GMP areespecially well suited for use in beverages and semi-solid foods such aspudding. For example, GMP is soluble in acid with an isoelectric pointof below 3.8, forms gels or foams, and has good heat stability. GMPactually enhanced the chocolate flavor used in the beverage with theadded feature that the chocolate flavor helped to mask the dairy flavorof GMP. These data suggest that GMP can be used to make a beverage thatis more palatable than the amino acid formulas currently required as theprimary source of protein in the PKU diet.

Example 3: Case Study of Adult with PKU Following a Ten Week GNP Diet

This Example is a case report of a 29 year old male with PKU who usedGMP-based foods as his sole protein source for a ten week period. Thetest subject reported that GMP-based foods tasted better than thestandard amino acid formula, and his plasma levels of Phe were loweroverall for the ten weeks that he consumed the GMP-based diet.

Approval was granted by the Health Sciences Institutional Review Board,University of Wisconsin-Madison to conduct an outpatient study insubjects with PKU to evaluate the safety and acceptability of dietaryGMP. A 29-year-old. male PKU subject with a genotype of R261Q and R408Wwas studied. The subject adhered to the low-Phe diet from birth through12 years but was off diet during adolescence, which resulted in thedevelopment of spastic quadriparesis and a seizure disorder that wastreated with standard anticonvulsant therapy. The subject completed a15-week study comparing GMP with his usual prescribed amino acid formula(Phenylade and Amino Acid Blend; Applied Nutrition, Cedar Knolls, N.J.,USA) as the primary source of dietary protein.

The protocol consisted of a diet in which he consumed his usual aminoacid formula for the first 3 weeks and the last 2 weeks of the study.During the middle 10 weeks of the study, GMP food products chosen by thesubject replaced all of the amino acid formula and included: GMP orangesports beverage (28 oz/day; 28 g protein), GMP pudding (1.5 cups/day; 15g protein) and GMP snack bar (1 bar/day; 5 g protein). See Table 7 forthe nutritional composition of the GMP foods.

For six weeks of the study, weighed portions of food with preciselycontrolled Phe content were sent to the subject's home. The Phe contentof the diets was determined by analysis of selected foods for amino acidcontent and calculation of Phe content for the remaining foods matchedin quantity and packing lot in both diets (US Department of AgricultureARS (2005) USDA Nurient Database for Standard Reference, Release 18).For the remaining 9 weeks of the study, the subject purchased andweighed his own food using menus planned with the metabolic dietician.Although Phe intake was well-controlled for 15 weeks, the results forPhe concentrations in blood and plasma presented here are based on the 6weeks that food was provided to the subject.

TABLE 7 Nutritional composition of GMP food products^(a) Serving EnergyProtein Phe Product size [kJ (kcal)] (g) (mg) Orange sports drink 340 g(12 oz) 419 (100) 12 33 Chocolate beverage 227 g (8 oz) 628 (150) 10 33Caramel beverage 227 g (8 oz) 670 (160) 10 26 Strawberry pudding 114 g(½ cup) 921 (220) 5 13 Chocolate pudding 114 g (½ cup) 921 (220) 5 21Ranch dressing 45 g (1.5 oz) 419 (100) 5 15 Cinnamon crunch bar 55 g (1bar) 711 (170) 5 14 ^(a)GMP food products were developed in theWisconsin Center for Dairy Research except for the ranch dressing andcinnamon crunch bar, which were developed by Cambrooke Foods of Boston.

The macronutrient profile provided by the amino acid and GMP diets wasconstant and included: 10 880-11 300 kJ/day (2600-2700 kcal/day), 10-11%energy from protein (0.84 g protein/kg), 24-26% energy from fat, and63-66% energy from carbohydrate. The subject maintained a body weight of87 kg during the study. The daily Phe content was 1100 mg Phe for 4weeks and 1180 mg Phe for 2 weeks; this provided approximately 13 mg Pheper kg of body weight. The amino acid formula and GMP food products eachprovided 0.6 g protein per kg body weight. The GMP food products weresupplemented to provide 130%, or 150% for tyrosine, of the amino acidscoring pattern for a complete protein for the following limiting aminoacids, expressed as mg amino acid per g GMP protein: histidine, 23;leucine, 72; tryptophan, 9; and tyrosine, 71. A multivitamin/mineralsupplement, a calcium/phosphorus supplement and 500 mg of L-tyrosinetwice a day were taken with the GMP diet to ensure intakes similar tothose provided by the amino acid formula.

Blood samples were obtained after an overnight fast and prior tobreakfast for determination of Phe concentration using one of twoanalytical methods known to give different values (Gregory et al (2007)Genet Med 9:761-765). Tandem mass spectroscopy (MS/MS) was used foranalysis of Phe concentrations in blood spots collected on filter paperby the subject between 09:00 and 09:30 (Rashed et al. (1995) Pediatr Res38: 324-331) and a Beckman 6300 amino acid analyzer was used foranalysis of the plasma amino acid profile obtained by venepuncture in alocal clinic between 12:00 and 12:30 (Slocum and Cummings (1991), AminoAcid Anaylsis of Physiological Samples, in Hommes, F A, ed., Techniquesin Diagnostic Human Biochemical Genetics: A Laboratory Manual. New York:Wiley-Liss, 87-126).

Statistical differences in blood and plasma amino acid concentrationsfor the amino acid and GMP dietary periods were evaluated by t-test withthe assumption that the amino acid measurements were independent overtime; p<0.05 was considered significant. When expressed relative to 100mg of Phe intake based on provision of meals with known Phe content,mean fasting plasma and blood Phe concentrations were significantlyreduced by 13-14% with consumption of the GMP diet compared with theamino acid diet (FIG. 5). The absolute concentration of Phe in plasmawas reduced by approximately 10% (from 736 to 667 mmol/L) withconsumption of the GMP diet compared with the amino acid diet (see Table8). Plasma tyrosine concentration was not significantly different withthe GMP and amino acid diets. No adverse effects were noted withconsumption of the GMP diet based on physical examinations and theresults of chemistry panel analyses which included electrolytes,albumin, prealbumin, and liver function tests.

Consistent with the amino acid profile of GMP and studies in the PKUmouse (see Example 1), significant increases in plasma concentrations ofthe LNAA were noted with ingestion of the GMP diet compared with theamino acid diet (Table 8). Threonine increased 2.6-fold, isoleucineincreased 1.7-fold, and the sum of the branched-chain amino acidsincreased 16% with the GMP diet. Interestingly, this subject waspreviously given a supplementation trial with a mixture of indispensableLNAA (PreKUnil; NiLab, Korsoer, Denmark), but this was stopped becausehis seizures became worse. The LNAA preparation contained a largerproportion of amino acids from tyrosine and tryptophan andproportionately less from threonine and the branched-chain amino acidsthan GMP, which contains approximately 80% of indispensable amino acidcontent from a combination of threonine and the branched-chain aminoacids. Thus, GMP appears to provide a safe dietary source of LNAA forthis subject. Plasma concentration of proline increased 40% with the GMPdiet in association with a 2-fold increase in proline ingestion with GMPcompared with the amino acid formula. There was a small ease in plasmaconcentration of glutamine and citrulline with the GMP diet comparedwith the amino acid diet that was not associated with a change in liverfunction tests.

TABLE 8 Fasting concentrations of amino acids in plasma with ingestionof the amino acid diet compared to the GMP diet in a single PKUsubject^(a) Concentration (imol/L) Amino acid Amino acid diet GMP dietNormal range Alanine 296 ± 26 331 ± 5  177-583  Arginine 58 ± 2 62 ± 315-128 Citrulline 26 ± 2  48 ± 7* 12-55  Cystine 31 ± 1 42 ± 5 5-82Glutamate  46 ± 10 53 ± 7 10-131 Glutamine 698 ± 22  858 ± 16** 205-756 Glycine 428 ± 7  428 ± 22 151-490  Histidine 69 ± 3 75 ± 2 41-125Isoleucine 42 ± 3  69 ± 1** 30-108 Leucine 92 ± 3 91 ± 3 72-201 Lysine189 ± 8  201 ± 5  48-284 Methionine 19 ± 1 19 ± 3 10-42  Ornithine 61 ±7 51 ± 6 48-195 Phenylalanine 736 ± 13  667 ± 24^(b) 35-85  Proline 153± 7   214 ± 13** 97-329 Serine 95 ± 1 88 ± 8 58-181 Taurine 65 ± 6  59 ±13 54-210 Threonine 95 ± 4  246 ± 12** 60-225 Tryptophan 33 ± 2 35 ± 210-140 Tyrosine 53 ± 5 45 ± 6 34-112 Valine 241 ± 15 275 ± 14 119-336 BCAA^(c) 374 ± 17  435 ± 15* 221-645  *,**Different from the AA diet, *p< 0.05, **p < 0.01. ^(a)Values are mean ± TSE, n = 4; blood samples wereobtained after an overnight fast and prior to breakfast between 12:00and 12:30 on different days distributed across the 5 weeks of the aminoacid diet and the 10 weeks of the GMP diet. ^(b)p = 0.073. ^(c)BCAA =sum of leucine, isoleucine, and valine.

Overall, the subject enjoyed the GMP diet and reported that he felt morealert with that diet than with his usual amino acid diet. Because thesubject enjoyed the GMP food products, he was more inclined todistribute them throughout the day. He consumed GMP food productsapproximately three times per day compared with once per day for theamino acid formula. It is well known that spacing the amino acid medicalfoods for PKU throughout the day lowers blood Phe levels owing toimproved utilization of amino acids for protein synthesis. Thus, oneexplanation for the reduced blood Phe levels with consumption of GMP isimproved protein synthesis due to consumption of a sufficient amount ofhigh-quality protein throughout the day. Alternatively, the greaterintake of LNAA with GMP may have contributed to the decrease in plasmaPhe levels, particularly the high intake of threonine (˜70 mg per kg perday).

In sum, compliance with the highly restrictive, low-Phe diet requiredfor the management of PKU remains poor during adolescence and adulthood,resulting in elevated blood Phe levels, neuropsychologicaldeterioration, and the tragic consequences of maternal PKU. Dietary GMP,an abundant food ingredient naturally low in Phe content, provides aninnovative approach to improving the nutritional management of PKU. ThisExample indicates that incorporation of low-Phe foods and beverages madewith GMP into the PKU diet improves the taste, variety and convenienceof the diet. A more tasty and versatile low-Phe diet may lead toimproved dietary compliance, metabolic control and ultimately quality oflife for individuals with PKU.

Example 4: Eleven Subject Clinical Trial Comparing GMP Supplemented withLimiting Amino Acids to Amino Acid Formula as Primary Protein Source forNutritional Management of PKU

This example shows that a diet using GMP supplemented with limitingamino acids as a protein source is a safe and highly acceptablealternative to synthetic AA formulas in the nutritional management ofPKU. As an intact protein source, GMP improves protein retention andphenylalanine utilization compared with AAs.

To further evaluate the potential benefits of GMP in the PKU diet, an8-d clinical investigation was conducted in individuals with PKU. Theobjective was to investigate the effects of substituting GMP foodproducts for the AA formula on acceptability, safety, plasma AAconcentrations, and measures of protein utilization in subjects withPKU.

Subjects and Methods

Subjects.

Twelve subjects with PKU who were routinely monitored at the BiochemicalGenetics Program, the Waisman Center, the University ofWisconsin-Madison, participated in this study between March 2006 andJune 2008. One subject (age: 10 y) withdrew from the study because shewas unable to complete the protocol. Thus, data from 11 subjects (agerange: 11-31 y; 7 males and 4 females) are reported (See Table 9). TheUniversity of Wisconsin-Madison Health Sciences Institutional ReviewBoard approved this study.

Criteria for participation included a diagnosis of classical or variantPKU and a willingness to consume≥50% of the prescribed volume of the AAformula. Optimal control of plasma phenylalanine concentrations,however, was not a prerequisite for participation. Optimal controlincludes maintenance of phenylalanine concentrations between 120 and 360μmol/L for neonates through age 12 y, between 120 and 600 μmol/L foradolescents, and <900 μmol/L for adults. The diagnosis of PKU was basedon concentration of phenylalanine measured before initiation of dietarytreatment during infancy; those with classical PKU show phenylalanineconcentrations of ≥1200 μmol/L (see Table 9). All subjects in this studywere diagnosed with classical PKU, except for one subject who wasdetermined to have a variant form of PKU (subject 1).

Mutation analysis was completed for each subject by DNA sequencing ofthe PAH gene (Laboratory Service Section, Texas Department of StateHealth Services, Austin, Tex.) using primers designed by Guldberg et al.(Hum Mol Genet. 1993; 2:1703-7). All subjects were compound heterozygousfor PAH mutations (Table 9). Five subjects showed 2 copies of mutationsconsidered to express primarily a classical phenotype and 6 subjectsshowed a classical mutation and a mutation observed in PKU patients withvariant and/or non-PKU hyperphenylalaninemia mutations.

Because a formal evaluation of each subject's dietary prescription hadnot been completed within 2 y of study recruitment, a phenylalanineallowance for all subjects was determined before study initiation. Forthis study, phenylalanine allowance was defined as the amount of dietaryphenylalanine intake that allowed for a constant plasma phenylalanineconcentration (±5% variance) as determined by sequential increases inphenylalanine intake with frequent monitoring of blood phenylalanineconcentrations in blood spots. Each subject's dietary phenylalanineallowance was verified by completing one or more “dry runs” in which allfood, beverages, and formula were provided for a 5-d period withmeasurement of phenylalanine concentrations in blood spots before and atthe end of each dry run. Plasma phenylalanine concentrations at theinitiation of the study ranged from 192 μmol/L (subject 10) to 1011μmol/L (subject 2; Table 9). To maintain these plasma phenylalanineconcentrations, the dietary phenylalanine allowance for the subjectsranged from 5.8 mg/kg (subject 10) to 26.7 mg/kg (subject 2).

TABLE 9 Individual characteristics of 11 subjects with phenylketonuriaPhenylalanine Age at concentration Baseline Dietary Dietary study Height/ at Age at plasma phenylalanine phenylalanine Subject initiation weightdiagnosis′ diagnosis′ phenylalanine² allowance per allowance per no./sexy cm/kg μmo/L d Mutation μmol/L mg mg 1/M 27 173/73 1270 35 R408W 6401151 15.8 IVS12nt1g→a 2/M 29 170/67 2208 15 R408W 1011 1793 26.7 R261Q3/M 14 164/52 1210 8 R408W 1009 673 13.0 IVS1Ont-11g→a 4/M 11 137/352051 7 IVS4nt5g→t 767 372 10.7 IVS12nt1g→a 5/M 12 148/45 2154 7 R408W690 979 21.6 Y356N 6/F 23  94/51 1488 10 R408W 536 545 10.6 IVS12nt1g→a7/F 28 159/64 2632 11 R408W 603 545 8.3 L242F 8/M 27 170/76 1924 11R261Q 810 971 12.8 E280K 9/F 20  93/64 1016 10 L48S 331 378 5.9 F299C10/F 31 157/70 1876 15 R408W 192 408 5.8 F299C 11/M 28 180/91 3122 13IVS1nt5g→t 392 711 7.8 IVS12nt1g→a ¹Age and phenylalanine concentrationsat diagnosis represent values when diet treatment was initiated duringinfancy. ²Values represent concentrations of phenylalanine in plasma 2.5h after eating breakfast on day 3 while consuming the prescribed aminoacid diet.

Study Protocol.

Each subject served as his or her own control in this metabolic study,which included 2 dietary treatments of 4 d each: the AA diet (days 1-4)and the GMP diet (days 5-8). One 24-h menu was designed for the AA dietand another for the GMP diet; the same menu was repeated on all days ofeach diet treatment (Table 10). In each diet, the AA formula or GMPproducts were divided equally in each of 3 meals during the day.Distribution of protein equivalents throughout the day improves proteinutilization and can lower plasma phenylalanine concentrations. Duringthe study, all food, beverages, snacks, formula, and GMP products wereweighed in grams by trained dietary staff at the Waisman Center or theUniversity of Wisconsin Clinical and Translational Research Core(UW-CTRC). To ensure identical intake on all days of the study, subjectswere encouraged to consume all foods and beverages. No subject failed todo this.

TABLE 10 Comparison of typical menus for the amino acid (AA) and theglycomacropeptide (GMP) diets¹ AA diet GMP diet Breakfast (103 mgphenylalanine, 14 g protein) Breakfast (102 mg phenylalanine, 14 gprotein) 177 mL PKU formula (0 mg phenylalanine) 296 mL GMP chocolatebeverage (51 30 g Cold cereal (51 mg phenylalanine) mg phenylalanine) 11g Pretzels (52 mg phenylalanine) 30 g Cold cereal (51 mg phenylalanine)Lunch (124 mg phenylalanine, 15 g protein) Lunch (124 mg phenylalanine,13 g protein) 177 mL PKU formula² (0 mg phenylalanine) 148 mL GMPchocolate beverage (24 mg 12.5 g Cinnamon toast (62 mg phenylalanine)phenylalanine) Cheese sandwich (45 mg phenylalanine; 64 g low 113 g (½cup) GMP chocolate pudding (38 protein bread) mg phenylalanine) 1 Slicelow-protein cheese, 8.7 g butter) Cheese sandwich (45 mg phenylalanine;64 g 125 g Peaches (17 mg phenylalanine) low-protein bread, 1 Slicelow-protein cheese, Dinner (220 mg phenylalanine, 18 g protein) 8.7 gbutter) 177 mL PKU formula² (0 mg phenylalanine) 125 g Peaches (17 mgphenylalanine) 9 g Bowtie pasta (53 mg phenylalanine) Dinner (226 mgphenylalanine, 18 g protein) Pasta Alfredo (61 mg phenylalanine; 60 glow- 1 GMP bar (33 mg phenylalanine) protein pasta, 237 mL GMP sportsbeverage (19 mg 5 g Regular bowtie pasta, 88 g low-proteinphenylalanine) Alfredo sauce) Pasta Alfredo (61 mg phenylalanine; 60 g92 g Broccoli, 50 g carrots and 14 g butter (105 low-protein pasta, mgphenylalanine) 5 g Regular bowtie pasta, 88 g low-protein 140 g Pears (7mg phenylalanine) Alfredo sauce) 237 mL Lemonade (0 mg phenylalanine) 92g Broccoli, 50 g carrots and 14 g butter (105 mg phenylalanine) 140 gPears (7 mg phenylalanine) 237 mL Lemonade (0 mg phenylalanine) ¹Allfoods were measured on a gram scale by trained staff. Since this studywas completed, improved recipes have been developed to further lower thephenylalanine content of all of the GMP products shown in this typicalmenu (12). For example, a GMP bar can now be produced with only 14 mgphenylalanine compared with 33 mg phenylalanine, and the GMP chocolatepudding now contains 21 mg phenylalanine compared with 38 mgphenylalanine in the original formulation used for this research. PKU,phenylketonuria. ²The PKU formula used in this menu is 40 g Phenex 2(Abbott Laboratories, Columbus, OH).

Each subject was provided with all food and formula to consume at homefor 2 d before initiation of the study and for days 1 and 2 of the AAdiet. Before dinner on day 2, each subject was admitted to the UW-CTRCfor continuation of the AA diet (days 3 and 4) and for 4 d of the GMPdiet (days 5-8). A physical exam was completed on all days of theUW-CTRC admission. All subjects were required to walk or completephysical activity 2-3 times/d to allow for an activity level consistentwith their usual routine. Timing of meals and snacks was also similar toeach subject's usual routine.

On days 1 and 2, each subject collected a blood spot on filter paper forphenylalanine and tyrosine analysis. During the UW-CTRC admission, bloodwas drawn daily for plasma AA and automated chemistry panel analysis tomeasure serum concentrations of prealbumin, albumin, total protein,electrolytes, glucose, blood urea nitrogen (BUN), creatinine, calcium,magnesium, phosphate, uric acid, total and direct bilirubin, alkalinephosphatase, and liver enzymes (γ-glutamyltranspeptidase, alanineaminotransferase, aspartate aminotransferase, and lactic dehydrogenase).All postprandial blood samples were drawn daily 3 h after the start ofbreakfast or 2.5 h after eating breakfast (days 3-8).

After the first 5 subjects had completed the protocol, the Data Safetyand Monitoring Board evaluated the protocol and study progress. As aresult of the board's suggestions, blood draws for chemistry panels wereeliminated on the first 2 d of the GMP diet (days 5 and 6), and anadditional fasting blood sample was added before breakfast on the last 2d of the AA diet (days 3 and 4) and on the last 2 d of the GMP diet(days 7 and 8) for the remaining 6 subjects. All fasting samples wereanalyzed for plasma AAs. The mean age of the 6 subjects for whom bothfasting and postprandial blood samples were obtained was 26±2 y andincluded 4 females and 2 males (subjects 6-11; Table 9).

Because the GMP food products were not supplemented with vitamins andminerals, all subjects were given a complete multivitamin with mineralsupplement (Phlexy-Vits; Nutritia North America, Gaithersburg, Md.) or acombination of Theragran M (Walgreen Co, Deerfield, Ill.) andTarget-Mins (Country Life, Hauppauge, N.Y.) during the GMP diet. Anysubject consuming a formula or formulas that did not contain vitaminsand minerals was given the same supplements provided for the GMP dietduring the AA diet. Additional calcium was given, if needed, to meetDietary Reference Intake (DRI) recommendations for age (Institute ofMedicine, Dietary Reference Intakes for Energy, Carbohydrates, Fiber,Fat, Protein and Amino Acids, Washington D.C.: National Academy Press,2002).

Study Diets.

GMP (Bio-Pure GMP; Davisco, Le Sueur, Minn.) was analyzed for AA contentat the University of Missouri Experimental Station Chemical Laboratory.The phenylalanine content for the commercial GMP was 0.4 gphenylalanine/100 g GMP with a protein content of 86.0 g/100 g GMP. ThisGMP was used for 3 subjects with a higher phenylalanine tolerance. For 9subjects with a lower phenylalanine tolerance, the original stock of GMPwas further purified to reduce the phenylalanine content to an averageof 0.21±0.01 g phenylalanine/100 g GMP with an average protein contentof 75.0±0.7 g/100 g GMP. Purification of the GMP decreased only thephenylalanine content; the proportion of the other AAs remainedunchanged in the purified GMP compared with the commercial GMP.

The GMP was supplemented with 4 limiting AAs, expressed as the finalconcentration in milligrams AA per gram GMP protein: histidine, 23;leucine, 72; methionine, 28; and tryptophan, 9. This is equivalent to130% of estimated needs on the basis of the 2002 DRIs (Institute ofMedicine, Dietary Reference Intakes for Energy, Carbohydrates, Fiber,Fat, Protein and Amino Acids, Washington D.C.: National Academy Press,2002). Because tyrosine is an indispensable AA in PKU, tyrosine wassupplemented at 150% of estimated needs for a final concentration of 71mg/g GMP protein. For the GMP diet, no attempt was made to duplicate theconcentration of supplemental tyrosine found in the various formulasconsumed by each subject because, in most cases, the tyrosine content ofthe formula was substantially greater than the estimated needs. Thus,for all subjects, tyrosine intake in the AA diet was greater thantyrosine consumed when GMP products were substituted.

Low-phenylalanine food products made with GMP as the protein source weredeveloped for this study by the Wisconsin Center for Dairy Research, theUniversity of Wisconsin-Madison. Before initiation of the study, eachsubject tasted a variety of food products made with GMP and selected 2to 3 products that would be included in menus for the GMP diet. GMPbeverages and foods included an orange-flavored sports beverage, achocolate-flavored or caramel-flavored beverage, chocolate or strawberrypudding, and a cinnamon crunch bar (as in Example 3; see Table 7). Therange of phenylalanine content in the GMP food products varied with thepurity of the GMP and the additional ingredients used to produce thesefoods and beverages, but, in general, a serving of GMP food productsprovided 5-10 g protein and 15-30 mg phenylalanine.

Diet Composition.

The AA and GMP diets were calculated on the basis of a prestudyevaluation of each subject's phenylalanine allowance and were controlledfor energy, protein, phenylalanine, and fat (see Table 11). The AA diet(days 1-4) included a subject's usual AA formula, which was differentfor each subject. For the GMP diet (days 5-8), GMP products weresubstituted for a subject's entire daily intake of AA formula. Thephenylalanine content of foods used to plan the menus was determined byAA analysis of selected foods and by calculation of phenylalaninecontent for the remaining foods. Foods that were not analyzed werematched in quantity, brand, and packing lot in both diets, whereas foodsanalyzed for phenylalanine content were used in variable amounts toaccount for the phenylalanine content of the GMP products.

Because of the limitations in data to quantitate the phenylalaninecontent of foods, dietary composites were collected for phenylalanineanalysis to verify calculations of phenylalanine content. Thus, aduplicate of all food, formula, and GMP food products consumed by eachsubject during a 24-h period was collected for 2 d during both the AAdiet and the GMP diet. Each duplicate was ground and freeze-dried, andan aliquot of each composite was sent to the University of Missouri forAA analysis. When comparing the composite analyses for each subject,phenylalanine content in the AA diet and the GMP diet was notsignificantly different (P=0.061).

TABLE 11 Nutrient composition of amino acid (AA) and glycomacropeptide(GMP) diets¹ Energy (kcal/kg) AA diet GMP diet <18 y old 56 ± 6 57 ± 5≥18 y old 35 ± 1 35 ± 2 Energy from protein (%)² 11 ± 1 10 ± 1 Energyfrom fat (%)³ 24 ± 1 23 ± 1 Phenylalanine intake (mg · kg⁻¹ · d⁻¹) 13 ±2 13 ± 2 Tyrosine intake (mg · kg⁻¹ · d⁻¹) 85 ± 9  51 ± 54 ¹Values aremeans ± SEMs and are based on calculated dietary intake; n = 11.²Protein from synthetic AAs represents 75% of the total protein in theAA diet and only 10% of the total protein in the GMP diet (from supple 

 menting the GMP with limiting indispensable AAs). All other protein inthe AA and GMP diets is from natural sources of intact protein. ³Totalfat intake ranged from 18% to 31% of total energy. A low fat intake istypical in those with phenylketonuria, given their selection ofcarbohydrate-based foods and the low fat content of many AA formulasdesigned for older individuals with this disorder (28). ⁴Significantlydifferent from the AA diet, P < 0.0001 (paired t test, pairing onsubject).

Measurements.

The blood spots collected by each subject to establish theirphenylalanine allowance and on prestudy days 1 and 2 were analyzed forphenylalanine and tyrosine by tandem mass spectrometry (MS/MS; data notshown). An AA profile was completed on all fasting and postprandialplasma samples collected on days 3-8 by using a Beckman 6300 amino acidanalyzer (Beckman-Coulter Inc, Fullerton, Calif.) equipped with an ionchromatography system that uses postcolumn ninhydrin derivatization. Thesamples were deproteinized with sulfosalicylic acid, centrifuged(14,000×g; 5 min) and passed through a 0.2-μm syringe filter beforeadding an internal standard and injecting it into the column.

Serum chemistry profiles were analyzed by using standard techniques atthe Clinical Laboratory, the University of Wisconsin-Madison Hospitaland Clinics. Plasma insulin was measured in postprandial samples byusing an radioimmunoassay specific for human insulin (Linco Research, StCharles, Mo.) on samples pooled within subjects for days 3+4 and days7+8. Insulin-like growth factor I (IGF-I) was measured in postprandialplasma samples for days 4 and 8 after removal of IGF-binding proteins byHPLC; the recovery of IGF-1 was 85-90%.

Statistical Analysis.

All statistical analysis was conducted with the statistical program Rfor Mac OS X version 1.12 (R Project for Statistical Computing,Wirtschaftsuniversität, Vienna, Austria). After dietary composites wereanalyzed, AA values within each diet were averaged for each subject(n=2), and then values between both diets were compared by using pairedt tests. Also, paired t tests, pairing on subject, were conducted tocompare plasma AA values from the last day of the AA diet (day 4) to thelast day of the GMP diet (day 8) for both postprandial and fastingsamples. Changes in the chemistry panel and liver function tests werecompared by using the same method. In addition, paired t tests wereconducted to compare fasting and postprandial AA concentrations withineach diet in the subset of 6 subjects from whom fasting plasma wasavailable. All comparisons were considered statistically significant ifP≤0.05. On the basis of the primary endpoint comparing plasmaphenylalanine concentration on the last day of the AA diet (day 4) withthe last day of the GMP diet (day 8), the achieved sample size (n=11)was sufficient to provide 80% power at P=0.05 if the change in plasmaphenylalanine concentration was 150 μmol/L.

Results

Diet Acceptability and AA Composition.

After consuming the GMP diet for 4 d, 10 of 11 subjects claimed that theGMP products were superior in sensory qualities to their usual AAformula. Moreover, at the conclusion of the study, 6 of the 7 adultsubjects expressed a strong preference to consume GMP products ratherthan their usual AA formula, if GMP became available to them as adietary option.

Compared with current recommendations, the analyzed intake (mg aminoacid/g of dietary protein) of all indispensable AAs met requirements forboth the AA and GMP diets (World Health Organization, Protein and AminoAcid Requirements in Human Nutrition, Geneva Switzerland: United NationsUniversity, 2007). However, AA analysis of the dietary compositesindicated several significant differences in AA intake with ingestion ofthe AA diet compared with the GMP diet (see Table 12). Because GMPcontains a high concentration of the LNAAs threonine and isoleucine,mean intakes of both of these AAs were significantly higher with the GMPthan the AA diet. Despite supplementation of GMP with tyrosine at 150%of DRI and leucine, histidine, tryptophan, and methionine at 130% of theDRI, the intake of these AAs, with the exception of methionine, wassignificantly lower with the GMP than the AA diet. The intakes of otherAAs that were significantly lower with ingestion of the GMP dietcompared with the AA diet included the indispensable AA lysine and thedispensable AAs arginine, alanine, glycine, and taurine.

TABLE 12 Analyzed profile of amino acids (AAs) from 24-h composites ofAA and glycomacropeptide (GMP) diets¹ AA diet GMP diet P value² AA gamino acid/24-h diet Alanine 4.08 ± 0.32 3.15 ± 0.07 0.039 Arginine 4.07± 0.23 0.80 ± 0.09 <0.0001 Aspartic acid 6.09 ± 0.37 5.08 ± 0.25 0.059Cysteine  143 ± 0.09 0.50 ± 0.05 <0.0001 Glutamic acid 10.3 ± 0.90 11.6± 0.64 0.219 Glycine 3.52 ± 0.26 0.99 ± 0.07 <0.0001 Histidine 1.89 ±0.10 1.27 ± 0.09 0.001 Isoleucine 3.70 ± 0.16 4.75 ± 0.26 0.004 Leucine6.51 ± 0.35 4.12 ± 0.28 0.0001 Lysine 4.53 ± 0.21 3.09 ± 0.17 <0.0001Methionine 1.24 ± 0.06 1.28 ± 0.08 0.605 Phenylalanine 0.79 ± 0.09 0.74± 0.08 0.061 Proline 5.27 ± 0.27  591 ± 0.32 0.198 Serine 3.27 ± 0.263.30 ± 018  0.883 Taurine 0.54 ± 0.08 0.25 ± 0.03 0.019 Threonine 3.00 ±0.10 7.12 ± 0.41 0.0001 Tryptophan 1.07 ± 0.07 0.57 ± 0.05 <0.0001Tyrosine 4.40 ± 0.17 2.63 ± 0.21 0.0001 Valine 4.50 ± 0.14 4.13 ± 0.210.105 BCAA 14.72 ± 0.62  13.00 ± 0.72  0.034 ¹Values are means ± SEMs; n= 22. BCAA, sum of leucine, isoleucine, and valine. ²Represents thedifference between the AA and the GMP diets by paired t test.

Physical Examination and Blood Chemistry.

There were no physical concerns detected on exam or expressed by anysubject to indicate any negative effect on health status when subjectsconsumed GMP as the primary protein source for a 4-d period. There wereno significant differences among serum concentrations of albumin,prealbumin, or total protein as indicators of protein status orcreatinine as an indicator of renal status measured on the last day ofthe AA diet (day 4) compared with the GMP diet (day 8; Table 13).However, BUN as an indicator of hepatic ureagenesis was significantlylower with ingestion of the GMP diet on both day 7 and day 8 than withthe AA diet on day 4 (see FIG. 6). Plasma concentration of IGF-I was notsignificantly different with the AA and GMP diets, which suggestsadequate protein nutrition in both diets. Plasma insulin concentrationwas higher and marginally significant with the GMP diet compared withthe AA diet (P=0.053), and serum glucose concentration was notsignificantly different. Serum carbon dioxide content, which isprimarily bicarbonate, was significantly higher with the GMP dietcompared with the AA diet, which is consistent with a lower systemicacid content. The mean concentrations of other standard chemistries,including electrolytes and liver function tests, remained within thenormal range with both diets (data not shown). The exception waselevated concentrations of various liver function tests (alanineaminotransferase and T-glutamyltranspeptidase) measured in subject 2,who was on anticonvulsant medications for his seizure disorder. However,further increases in these liver function tests were not detected withingestion of the GMP diet compared with the elevations measured atadmission to the study.

TABLE 13 Effect of amino acid (AA) and glycomacropeptide (GMP) diets onpostprandial indexes of protein and glucose metabolism¹ Test AA diet GMPdiet P value² Blood urea nitrogen (mmol/L) 4.2 ± 0.3  3.4 ± 0.2  0.02Creatinine (itmol/L) 73 ± 5.5 73 ± 4.6 1.00 Total protein (g/L) 68 ± 1.467 ± 1.4 0.27 Albumin (g/L) 44 ± 0.9 44 ± 0.8 0.84 Prealbumin (g/L) 317± 7.5  310 ± 7.3  0.22 Insulin-like growth 13.5 ± 1.3  13.7 ± 1.5  0.14factor I (nmol/L) Insulin (pmol/L) 84 ± 22  116 ± 34  0.05 Glucose(mmol/L) 4.5 ± 0.1  4.8 ± 0.1  0.14 CO₂ content (mmol/L) 26 ± 0.6 28 ±0.6 0.01 ¹Values are means ± SEMs; n = 11, except total protein andinsulin for which n = 10; all values are within normal range. Values arefor serum except those for insulin-like growth factor I and insulin,which used plasma. ²Difference between the last day of the AA diet (day4) and the GMP diet (day 8) by paired t test, pairing on subject.

Plasma AA Concentrations.

The concentration of total AAs in plasma was significantly greater, andthe concentration of BUN was significantly lower, with the GMP dietcompared with the AA diet when measured 2.5 h after eating breakfast(see FIG. 6). This is consistent with slower absorption of AAs from anintact source of protein compared with synthetic AAs and higher insulinconcentrations with ingestion of GMP.

Phenylalanine and Tyrosine.

There was no significant difference (P=0.173) in the mean postprandialconcentration of phenylalanine in plasma with ingestion of the AA diet(day 4) compared with the GMP diet (day 8; FIG. 7). The mean change inthe concentration of phenylalanine in plasma was 57±52 μmolphenylalanine/L. Among individual subjects, the response of plasmaphenylalanine concentration to ingestion of the GMP diet washeterogeneous, ranging from a decrease of 175 μmol phenylalanine/L to anincrease of 257 μmol phenylalanine/L. Overall, there was no consistentassociation between a change in the concentration of phe in plasma withingestion of the AA diet compared with the GMP diet and sex, genotype,and age.

Concentrations of phenylalanine in both fasting and postprandial plasmawere available on day 4 (AA diet) and on day 8 (GMP diet) for a subsetof 6 adult subjects. The postprandial response to the GMP diet was notsignificantly different with this subset (n=6) than with the first 5subjects. Ingestion of the AA diet for 4 d resulted in a significant 10%increase in the concentration of phenylalanine in plasma obtained afteran overnight fast compared with the concentration of phenylalanine inpostprandial plasma obtained 2.5 h after eating breakfast (P=0.048; seeFIG. 8). In contrast, ingestion of the GMP diet for 4 d resulted in nosignificant change in the concentration of phenylalanine in plasma whencomparing plasma obtained in a fasting state to plasma obtained in apostprandial state.

Tyrosine is an important AA in the PKU diet because it is indispensableand a precursor of adrenaline, norepinephrine, melanin, and thyroxine.Concentrations of tyrosine in plasma obtained in the postprandial orfasting samples were not significantly different with ingestion of theGMP or AA diets (see Table 14). Concentrations of tyrosine in plasmaafter an overnight fast were decreased compared with postprandialconcentrations with ingestion of both the GMP and the AA diet; however,the GMP diet resulted in a mean fasting tyrosine concentration that wasbelow the normal range.

Additional AAs.

The most dramatic change in the profile of AAs in plasma with ingestionof the GMP compared with the AA diet was the 2.25- to 2.47-fold increasein postprandial concentrations of the nontoxic LNAA isoleucine andthreonine, which places these values above the normal clinical range(see Table 14). A significant increase in plasma concentration ofisoleucine and threonine with the GMP diet occurred within 24 h ofingesting the GMP diet and was consistent with the high concentrationsof these AAs in GMP (See FIG. 9). However, there was no furthersignificant increase in plasma concentration of isoleucine and threonineafter days 5 and 7, respectively. The concentration of isoleucine wasnot different in plasma obtained after an overnight fast, whereas theconcentration of threonine in fasting plasma remained ˜2-fold greaterwith ingestion of the GMP compared with the AA diet.

Consistent with the AA profile of the GMP and AA dietary composites,there were significantly lower postprandial concentrations of ornithineand tryptophan in plasma and significantly higher concentrations ofisoleucine and threonine in plasma with consumption of the GMP comparedwith the AA diet. After an overnight fast, plasma concentration ofarginine was significantly lower and concentration of threonine wassignificantly higher with the GMP compared with the AA diet (see Table14).

TABLE 14 Effect of amino acid (AA) and glycomacropeptide (GMP) diets onfasting and postprandial (PP) concentrations of AAs in plasma¹ Responseto AA diet GMP diet diet fasting Fasting² Fasting² P compared with AAPP² μmol/L P value³ PP² μmol/L value′ PP P value⁴ Alanine 455 ± 52 356 ±25 0.029 514 ± 45 401 ± 50 0.001 0.743 Arginine  62 ± 14  57 ± 5 0.694 47 ± 5  47 ± 6 0.845 0.551 Citrulline  37 ± 4  26 ± 5 0.138  23 ± 3  26± 4 0.084 0.063 Cystine  43 ± 1  43 ± 2 0.899  37 ± 2  41 ± 3 0.0190.062 Glutamic acid  40 ± 8  50 ± 10 0.207  43 ± 10  49 ± 9 0.556 0.692Glutamine 635 ± 29 628 ± 15 0.747 659 ± 33 623 ± 28 0.025 0.095 Glycine415 ± 62 399 ± 55 0.473 346 ± 40 371 ± 47 0.099 0.112 Histidine  85 ± 9 78 ± 5 0.206  82 ± 6  75 ± 4 0.066 0.681 Isoleucine  57 ± 6  49 ± 40.352 119 ± 15  54 ± 5 0.015 0.004 Leucine 120 ± 20  96 ± 3 0.313  86 ±11  83 ± 4 0.823 0.264 Lysine 210 ± 19 181 ± 9 0.062 191 ± 24 171 ± 160.138 0.311 Methionine  24 ± 2  24 ± 1 0.832  31 ± 4  25 ± 3 0.150 0.144Ornithine  74 ± 7  60 ± 8 0.004  50 ± 4  62 ± 18 0.460 0.129Phenylalanine 462 ± 100 508 ± 95 0.048 472 ± 106 483 ± 101 0.349 0.037Proline 200 ± 15 144 ± 12 0.001 234 ± 37 160 ± 20 0.012 0.322 Serine 131± 21 122 ± 16 0.123 127 ± 14 120 ± 11 0.517 0.821 Taurine  73 ± 19  82 ±18 0.154  73 ± 13  63 ± 11 0.145 0.027 Threonine 158 ± 22 135 ± 17 0.013354 ± 44 265 ± 29 0.030 0.064 Tryptophan  48 ± 5  44 ± 3 0.269  34 ± 3 42 ± 2 0.048 0.006 Tyrosine  81 ± 8  38 ± 3 0.001  56 ± 10  29 ± 30.027 0.155 Valine 240 ± 9 194 ± 13 0.015 241 ± 21 187 ± 5 0.048 0.521BCAA 417 ± 28 347 ± 14 0.084 445 ± 45 324 ± 8 0.048 0.062 ¹Values aremeans ± SEMs; n = 6, except for cystine for which n = 5. BCAA, sum ofleucine, isoleucine, and valine. ²PP plasma concentrations of omithine(P = 0.019) and tryptophan (P = 0.003) were significantly lower, butwithin the normal range, and isoleucine (P = 0.003) and threonine (P =0.004) were significantly higher with ingestion of the GMP diet thanwith the AA diet and above the normal range. The only significantdifferences in fasting AA concentrations were a decrease in arginine (P= 0.008) and an increase in threonine (P = 0.001) with ingestion of theGMP diet compared with the AA diet. ³There was a significant effect oftime in the repeated-measures ANOVA. Statistical analysis by paired ttest, pairing on subject, is from data collected on the last day of theAA diet (day 4) and the last day of the GMP diet (day 8). ⁴The responseto a diet is calculated first by finding the difference between fastingand PP AA concentrations for each subject on the AA diet and on the GMPdiet and then by comparing the differences by paired t test, pairing onsubject.

This is the first clinical trial to investigate the efficacy ofsubstituting intact protein from GMP food products for synthetic AAformulas that are currently required for nutritional management of PKU.No adverse health problems were found, and blood chemistries remainednormal when subjects with PKU consumed GMP as their primary proteinsource for 4 d in this controlled metabolic diet study (Table 13).Furthermore, the GMP products were preferred by the subjects, whichconfirms the results of blind taste tests comparing GMP to AA productsin those with PKU (See Example 2). Thus, foods and beverages made withGMP are both safe and highly acceptable for use in thephenylalanine-restricted diet for PKU.

Over a period of 4 d, there was no significant change in plasmaphenylalanine concentrations with ingestion of GMP products comparedwith the AA formula (FIG. 7). GMP, as an intact protein source, maydelay absorption of AAs and improve utilization of phenylalanine andother AAs for protein synthesis when compared with a synthetic AAsource. In this study, the AA diet showed a significantly higher meanfasting phenylalanine concentration compared with the postprandialphenylalanine concentration (FIG. 8), whereas there was no significantdifference in fasting and postprandial phenylalanine concentrationsapparent with the GMP diet (Table 14). This suggests that the GMP dietinduced less variation and potentially lower mean concentrations ofphenylalanine in plasma over a 24-h period. This is consistent withincreased protein retention and decreased oxidation of AAs inassociation with a slower rate of absorption of AAs when the dietaryprotein source is an intact protein, such as GMP, as compared with afree AA source, such as AA formula.

Evidence of improved protein retention with the GMP diet was also shownby a lower serum BUN and higher plasma insulin and total AAconcentrations when measured 2.5 h after eating a breakfast containingGMP compared with one containing AAs (Table 13, FIG. 6). Urea isproduced linearly in response to plasma AA concentrations, and controlof nitrogen balance is primarily regulated by urea production. BUN, as ameasure of hepatic utilization of AAs for urea synthesis, would beexpected to remain lower with slower splanchnic AA release. Thus, aslower, more gradual and sustained elevation in plasma AA concentrationwith an intact protein source, in conjunction with a lower BUNconcentration, suggests that fewer AAs are degraded for urea productionand instead are retained for protein synthesis when GMP is substitutedfor synthetic AAs as the primary protein source.

Postabsorptive AAs, including isoleucine and threonine (Calbet et al., JNutr 2002; 132:2174-82), are known to stimulate insulin release withsubsequent stimulation of protein synthesis and inhibition of proteindegradation (Schmid, et al., Pancreas 1992; 7:698-704). Because GMPinduces a slower and more prolonged release of amino acids, the insulinresponse and stimulus of net protein synthesis may be potentiated. Inaddition, whey protein has been shown to increase insulin concentrationto a greater extent than other milk protein fractions or other intactprotein sources (Nilsson et al., Am J Clin Nutr 2004; 80:1246-53). Thus,the ability of GMP to slow AA catabolism and ureagenesis may reflectincreased postprandial concentrations of threonine and isoleucine actingas insulin secretagogues as well as delayed absorption of AAs.

For this study, GMP was supplemented with the following 5 limiting AAson the basis of the 2002 DRI recommendations: histidine, leucine,methionine, tryptophan, and tyrosine. Plasma concentrations ofhistidine, leucine, and tryptophan remained within the normal range,which suggests adequate supplementation of these AAs in the GMP diet(Table 14). In contrast, plasma concentration of tyrosine was below thenormal range when measured in the fasting state (Table 14), whichsuggests that additional tyrosine supplementation may be required inGMP. Indeed, additional tyrosine from a supplement providing 1000 mg/dallowed for plasma tyrosine concentrations to remain within the normalrange for one subject who consumed GMP as his primary protein source for10 wk (see Example 3). In summary, our data suggest that GMP must besupplemented with arginine, histidine, leucine, tryptophan, and tyrosineto provide a complete source of dietary protein in the PKU diet.

Lifelong adherence to the PKU diet is very difficult, often resulting inpoor compliance and the neuropsychological consequences ofhyperphenylalaninemia. This research shows a new, improved paradigm forthe PKU diet through the use of palatable foods and beverages made withthe intact, low-phenylalanine protein GMP instead of synthetic AAs. Whensupplemented with limiting indispensable AAs, GMP appears to be a safeand acceptable alternative to synthetic AAs as the primary proteinsource for nutritional management of PKU. As an intact protein, GMPdelays absorption of AAs and improves protein retention andphenylalanine utilization compared with a diet that provides themajority of nitrogen from AAs.

Example 5: Increasing GMP Purity and Using a Mass Balance Calculationfor Amino Acid Supplementation of GMP-Based Foods

This Example illustrates methods for increasing the purity of GMP usedin making GMP-based foods. In addition, the Example illustrates the useof a mass balance calculation for determining the extent ofindispensable amino acid supplementation of GMP-based foods.Introduction

Daily recommended intakes (DRI) of indispensable amino acids forindividuals older than 1 y have been established based on the nitrogencontent of foods (see Table 15).

TABLE 15 Daily recommended intake (DRI) of indispensable amino acids forchildren ≥1 y of age and all other age groups. (Adapted from Inst. ofMedicine 2005, Dietary Reference Intakes for Energy, Carbohydrate,Fiber, Fat, Fatty acids, Cholesterol, Protein, and Amino Acids,Washington, DC: Natl. Academies Press). Amino acid DRI mg/g N DRI mg/gPE ^(a) His 114 18 He 156 25 Leu 341 55 Lys 320 51 Met + Cys 156 25Phe + Tyr^(b) 291 47 Thr 170 27 Trp 43 7 Val 199 32 ^(a) PE = N × 6.25.^(b)Only Tyr was used to supplement GMP.

DRI values for amino acids are often reported on a protein equivalent(PE) basis defined as total nitrogen (N) times a conversion factor of6.25 (Inst. of Medicine 2005). Most proteins contain about 16% nitrogenand a conversion factor of 6.25 (100 g protein/16 g N=6.25 g protein/gN) is appropriate (Nielsen SS 2003, Food Analysis, 3rd ed. New York:Kluwer Academic/Plenum Publishers). However, the nitrogen to proteinconversion factor varies among foods. For example, the nitrogen toprotein conversion factor used for most dairy proteins is 6.38 (Id.).GMP is a heterogeneous peptide, some GMP molecules are glycosylated andothers are not. Because of this, the conversion factor for a GMPmolecule can range from 6.70 to 9.55 depending on the extent ofglycosylation present (Dziuba and Minkiewicz 1996, Int Dairy J6(11-2):1017-44). Ambiguity in the definition of protein mass isillustrated by one manufacturer of GMP that uses a conversion factor of6.47 to report protein and 7.07 to report GMP (Davisco Foods Intl.).

The actual protein content of foods is difficult to measure when theprecise nitrogen to protein conversion factors are unknown. Proteins maycontain nonprotein nitrogen, or basic amino acids, which are higher innitrogen than other amino acids. For GMP to provide the recommendedlevels of indispensable amino acids in the diet, the protein compositionof foods consumed must be known using an unambiguous definition of whatconstitutes 1 g of protein. In this Example, a conversion factor of 6.25was used to comply with the Inst of Medicine (Inst. of Medicine. 2005)definition of protein used to establish the DRI for indispensable aminoacids for individuals older than 1 y (see Table 15).

Current large-scale technologies to isolate GMP from whey use ionexchange chromatography or ultrafiltration. GMP has an isoelectric point(pI) below 3.8, whereas other major whey proteins have pI values above4.3. This physicochemical difference between GMP and other whey proteinsis commonly used in isolation processes to separate GMP from whey.Commercially available GMP isolated by ion exchange chromatography istypically not pure enough for PKU foods because it contains too much Phefrom residual whey proteins (i.e. 5 mg Phe/g product, manufacturerliterature, Davisco Foods Intl., Eden Prairie, Minn., U.S.A.).Traditional amino acid formula is free of Phe, which allows anindividual with PKU to consume natural foods that contain Phe to meettheir daily allowance. In order for GMP to be a feasible proteinreplacement for amino acid formula in the PKU diet, improved processesare needed to increase GMP purity and reduce Phe content.

To conduct a human clinical trial to test the safety and feasibility ofGMP foods for individuals with PKU, a pilot scale process was developedto prepare highly purified GMP sufficient to feed 15 individuals for 4d. Food-grade materials and food-approved facilities were utilized tomanufacture 5 kg of purified GMP using the following unit operations insequence (1) cation exchange chromatography, (2) ultrafiltration anddiafiltration (UF/DF), and (3) lyophilization. Furthermore, a massbalance calculation was developed to provide a clearly defined basis foramino acid supplementation. Purified, supplemented GMP was used toprepare GMP foods consumed in the human clinical trial.

Materials and Methods

This section has been divided into 3 subsections: (1) unit operationsused to manufacture purified GMP using food-grade materials, (2) massbalance calculations used to determine GMP recovery and the mass ofamino acids for supplementation of purified GMP, and (3) preparation andanalysis of a GMP food consumed in the clinical trial and 1 patientresponse.

GMP Purification Process

Contaminating whey proteins in crude GMP (BioPure GMP, Davisco FoodsIntl.) were trapped by adsorption onto a cation exchange resin and GMPwas collected in the flow-through fraction.Ultrafiltration/diafiltration (UF/DF) was used to concentrate the GMPand wash out peptides, salts, and nonprotein nitrogen. Lyophilizationwas used to dry the purified, concentrated GMP.

Cation Exchange Chromatography.

A 20-cm-dia chromatography column (INdEX, GE Healthcare, Piscataway,N.J., U.S.A.) was packed with SP Sepharose Big Beads (GE Healthcare).The column volume (CV) was 5.34 L, and the bed height was 18 cm.

Feed solution (75 g/L) was prepared by mixing crude GMP (BioPure GMP,Davisco Foods Intl.) with 10 mM sodium lactate, pH 4, and filteringthrough a 0.45-μm pore size filter (Sartobran P, Sartorius, Edgewood,N.Y., U.S.A.). Equilibration and elution buffers were food-grade 50 mMsodium lactate, pH 4, and 10 mM NaOH, pH 12, respectively. Equilibrationbuffer and GMP feed solution were held at 4° C. to inhibit microbialgrowth. Elution buffer was held at 22° C. Flow rate was 950 mL/min. Eachcation exchange cycle consisted of 4 steps: (1) the column was broughtto pH 4 using 2 CV of equilibration buffer, (2) 0.5 CV of feed solutionwas applied to the column, (3) the column was rinsed with equilibrationbuffer wherein the first 0.3 CV of effluent was discarded (column deadvolume) and the next 2 CV of purified GMP was collected, and (4) boundproteins were desorbed from the column using 2.5 CV of elution buffer.Each of the 5 campaigns consisted of 9 to 11 consecutive cycles. About100 L of dilute GMP protein solution were generated from each campaign.The cation exchange column was cleaned by pumping in 0.2 M NaOH, holdingfor 1 h, and then storing the column in 10 mM NaOH.

Ultrafiltration and Diafiltration.

GMP effluent from the cation exchanger was adjusted to pH 7 by additionof 1M NaOH, and concentrated at 60° C. using a hollow fiberultrafiltration membrane (3 kDa, 3.3 m², UFP-3-C-55, GE Healthcare). Theapplied pressure was 1.4 bar. GMP solution was concentrated from 100 to10 L, and then 20 L of distilled water added and the solution wasconcentrated again to 10 L. The concentrate was filtered using a 0.45-μmpore size filter (Sartorius) into a sanitized container and stored at 4°C. Before and after each use, the UF membrane was cleaned for 30 minusing 0.2 M NaOH containing 100 ppm NaOCl (bleach) at 50° C. The UFmembrane was stored in 10 ppm NaOCl.

Lyophilization.

Concentrated, sterile-filtered GMP solution was frozen into a thin layeronto 1.2 or 2 L glass lyophilization flasks and dried for 48 h(Lyphlock6, Labconco, Kansas City, Mo., U.S.A.). GMP powder wasrecovered, weighed, and a portion used for analysis.

Composition Analysis.

Crude protein analysis (CP) and complete amino acid profiling (AAP) wereconducted by the Experiment Station Chemical Laboratories (Univ. ofMissouri-Columbia, Columbia, Mo., U.S.A.). AOAC official method 982.30was used for AAP, and AOAC official method 990.03 was used for CP([AOAC] Assn. of Official Analytical Chemists, Intl. 2005. Officialmethods of analysis of official analytical chemists. 18th ed.Gaithersburg, Md.: AOAC). Results were reported as gram per 100 g dryweight of purified GMP. A conversion factor of 6.25 times total nitrogenwas used to express the results on a PE basis. Duplicate analyses wereperformed for all samples.

Mass Balance Calculations

Mass balance calculations are presented to describe (1) the calculationsused to determine GMP recovery from the manufacturing process, (2) thelysine basis used for amino acid supplementation, and (3) the methodused to determine the required amount of supplemental amino acids.

GMP Recovery.

Recovery was calculated on a PE basis. Grams of PE in the feed solution(M_(PE,feed)) were determined by multiplying the GMP feed concentration(75 g/L) by the grams of PE per gram of powder (from CP analysis) andthen multiplying by the total volume of feed solution processed. Thetotal grams PE recovered (M_(PE,recovered)) was obtained by multiplyingthe mass of purified GMP powder times the grams of PE per gram ofpurified GMP (from CP analysis). GMP recovery (%) was equal toM_(PE,recovered)/M_(PE,feed)×100.

Lysine Basis for Amino Acid Supplementation.

Amino acid supplementation is required for purified GMP to meet thenutritional targets set by the DRI and the clinical trial. Lysine (Lys)was chosen as the basis for the mass balance supplementationcalculation, because it was closest to the target value (see Table 16,column C compared with D). Only 5 indispensable amino acids for PKUrequired supplementation: His, Leu, Met, Trp, and Tyr. Dispensable andconditionally indispensable amino acids were not supplemented.

Free amino acids are absorbed and degraded faster than those provided byintact proteins. Therefore, the target amino acid composition for theclinical trial was set above the DRI levels. Targets for His, Leu, Met,and Trp were set at 130% of the DRI level. Tyr was supplemented at 150%of the DRI level, because amino acid formulas are often enriched withhigh levels of Tyr. In the tables and figures pertaining to thesupplementation of GMP to meet the DRI, Phe and Tyr are listed together,as are Met and Cys, because Tyr and Cys are conditionally dispensableamino acids that can be synthesized from Phe and Met, respectively.However, in a PKU patient, Tyr cannot be synthesized from Phe.

TABLE 16 Supplemental amino acid (AA) calculation method. E = D − C F =C + E G H = F × B/G Ignores PE of added Includes PE of added B D AA (notincluded in AA (included in A GMP C = A/B Clinical denominator)denominator) GMP AA PE^(A) g GMP trial AA Corrected Correctedcomposition^(A) PE/g composition target supplementation Supplemented PEg PE/g supplemented Amino mg/g purified purified on PE basis mg/grequired mg/g composition purified composition acid GMP GMP mg/g PE PEPE mg/g PE GMP mg/g PE His 1.15 ± 0.07 —  1.5 ± 0.1 23 22 23.5 ± 0.1^(a)—  21 ± 2^(a) Ile 75.5 ± 4.2 —  102 ± 2 25 0  102 ± 2^(a) —  90 ± 10^(a)Leu   17 ± 1 —   23 ± 0.2 72 49   72 ± 0.2^(a) —  66 ± 7^(a) Lys   44 ±3 — 60.1 ± 0.8 51 0 60.1 ± 0.8^(a) —  55 ± 6^(a) Met +   15 ± 2 — 20.5 ±0.5 33 12 32.5 ± 0.8^(a) —  30 ± 3^(a) Cys Phe +   3 ± 1 —  2.6 ± 0.6 7168 70.6 ± 0.6^(a) —  64 ± 6^(a) Tyr Thr  120 ± 10 —  161 ± 2 27 0  161 ±2^(a) — 150 ± 10^(a) Trp   0 ± 0 —   0 ± 0 9 9   9 ± 0^(a) —  8 ±0.8^(a) Val   62 ± 3 —   85 ± 2 32 0   85 ± 2^(a) —  78 ± 8^(a) PE —0.74 ± 0.05 — — — — 0.81 ± 0.06 — ^(A)Composition obtained by analysis.Values are mean ± SD. Sample size was n = 2. Same letters in column Fand H indicate no significant statistical difference (P > 0.05).

Amino Acid Supplementation Calculations.

A challenge with supplementation is that adding amino acids to thepurified GMP changes the amino acid target (mg amino acid/g PE) bychanging both the numerator (mg amino acid) and the denominator (g PE).Two methods will be illustrated for the supplementation calculation: oneincludes the change in the denominator and the other does not. Bothmethods account for the change in the numerator. The steps used tocalculate amino acid supplementation are presented in Table 16 above.The experimental results for AAP were used to obtain the milligram ofeach amino acid per gram of purified GMP (column A). The experimentalresults for CP (column B) were divided into column A to obtain the GMPamino acid composition on a PE basis (column C). Conversion to a PEbasis (column C) was needed to compare the purified GMP amino acidcomposition to the clinical trial target values (column D). The aminoacid values of purified GMP were subtracted from each of the clinicaltrial target values to yield the required mass of each supplementedamino acid (column E). By adding the required amino acid values (columnE) to the purified GMP amino acid values (column C), the composition ofthe supplemented GMP was calculated (column F). This method for thesupplementation calculation ignored the impact of adding amino acids onthe denominator of the amino acid composition (mg/g PE).

To account for the change in the denominator, the increase in the gramsof PE per gram of purified GMP due to supplementation must be taken intoaccount (Table 16, columns G and H). To do this, the total nitrogencontribution of the supplemental amino acids needed for 1 g of purifiedGMP was determined from the molecular formula and multiplied times 6.25to yield the total grams of PE contributed by the added amino acids. Thegrams of PE from the supplemented amino acids were added to the grams ofPE per 1 g of purified GMP (column B) to yield the corrected grams of PEper gram of purified GMP (column G). The supplemented composition(column F) was multiplied by column B and divided by column G to accountfor the change in the denominator (column H).

The corrected supplemented GMP composition was not statisticallysignificantly different from the uncorrected composition (Table 16,column H compared with F) (P>0.05). Therefore, the calculation methodused to supplement purified GMP in this study assumed that thecontribution of added amino acids to the denominator was negligible.

Preparation and Analysis of GMP Foods.

The recipe for GMP strawberry pudding is presented in Table 17. PurifiedGMP, supplemental amino acids, and nondairy creamer (Flavorite Non-DairyCreamer, SuperValu, Eden Prairie, Minn., U.S.A.) contributed amino acidsto the GMP strawberry pudding. Amino acid contribution from purifiedGMP, nondairy creamer, and added amino acids were used to calculate theamino acid composition of the pudding on a PE basis using the methodpresented previously in Table 16.

TABLE 17 GMP strawberry pudding recipe Ingredients Dry mix percent %(w/w) Purified GMP^(a) 12.94 Supplemented amino acids^(a) His 0.22 Leu0.70 Met 0.11 Tyr 0.68 Trp 0.09 Food ingredients Nondairy creamer^(a)39.73 Sucrose 32.22 Starch 8.60 Strawberries, dried 2.13 Citric acid1.59 Sodium chloride 0.57 Strawberry flavor 0.41 Red color 0.01 Dry mixtotal 100 ^(a)Purified GMP, supplemental amino acids, and nondairycreamer contribute amino acids to final product.

Statistical Analysis.

Statistical analysis was performed using a one-way analysis of variance(ANOVA) (Minitab Statistical Software, Release 13.32, State College,Pa., U.S.A.) to compare the composition of supplemented GMP with andwithout taking the denominator into account (Table 16). Confidenceintervals were constructed to compare the calculated composition of GMPstrawberry pudding to the observed composition and the DRI values. A 95%confidence level was used to construct confidence intervals, andstatistical significance was declared at α<0.05.

Results

The goal of this study was to produce purified GMP with reduced Phe inquantities great enough to supply 15 PKU subjects with GMP foods duringtheir participation in an 8 d clinical trial. Purified GMP requiredsupplementation with indispensable limiting amino acids to provide anutritionally complete protein source for use in GMP foods. The safetyand efficacy of GMP foods as a palatable source of protein for the PKUdiet was tested. The following sections discuss how these objectiveswere met and have been separated into the effect of the pilot plantprocess on the recovery and Phe content of purified GMP, the massbalance on amino acids in GMP strawberry pudding and comparison of theamino acid composition of the GMP strawberry pudding to the DRI and anamino acid formula, and the effect of the purified, supplemented GMPfoods on the plasma amino acid levels of PKU subjects.

Effect of Purification Process on Phe Content and GMP Recovery.

Table 18 contains the Phe content, recovery, and number of cationexchange cycles for each campaign. Phe in crude GMP was reduced 47% bythe purification process, from 4.7±0.5 mg/g PE to 2.7±0.4 mg/g PE.Average GMP recovery was 52±4%. The low recovery of GMP was attributedprimarily to a portion of GMP that bound to the cation exchange columnand was not recovered in the flow-through fraction. The purificationprocess used to increase the purity of commercially available GMP gaveconsistent, repeatable Phe concentrations, and there were no statisticaldifferences between Phe concentrations in purified GMP produced by the 5campaigns (P>0.05).

TABLE 18 Phe content of purified and commercially available GMP Phe^(a)GMP Campaign Cycles mg/g PE recovery % 1 10 3.3 ± 0.2 51 2 11 2.6 ± 0.255 3 10 3 ± 0 55 4 9 2.2 ± 0.5 53 5 9 2.7 ± 0.1 44 Average purified GMP— 2.7 ± 0.4 52 ± 4 Commercially available GMP — 4.7 ± 0.5 —^(a)Composition obtained by analysis. Values are mean ± SD Sample sizewas n = 2.

GMP transmission through the UF membrane for each campaign is presentedin Table 19. Overall, GMP retention by the UF membrane was 96±2%.Although GMP has a molecular weight of about 7 kDa, it has an apparentmolecular weight of 45 kDa at pH 4 and above. The pH of GMP solution wasincreased to 7 for the UF/DF step to minimize GMP transmission throughthe 3 kDa membrane. This likely explains the high recovery found for theUF step.

TABLE 19 GMP transmission through UF membrane GMP Campaign GMPtransmission (%) 1 3.4 2 6.8 3 2 4 3.6 5 2 Mean SD (n = 5) 4 ± 2

Comparison of Amino Acid Supplementation Calculations and GMP FoodComposition.

The GMP strawberry pudding was analyzed and the composition compared tothe calculated amino acid composition (Table 20). Purified GMP (Table20, column A) was supplemented with amino acids (column B), determinedusing the mass balance method that ignored the change in denominator dueto added amino acids; similarly for the added amino acids from thenondairy creamer (column C). The sum of columns A, B, and C was thecalculated composition of the GMP pudding ignoring changes in thedenominator (column D).

TABLE 20 Calculated and analyzed amino acid (AA) composition of GMPstrawberry pudding E B C D = A + B + C (Added AA Added AA Added AA(Added AA not included in A from from included in denominator) Purifiedsupplemented nondairy denominator) Corrected, F GMP ^(A) amino acids^(B) creamer ^(A) Calculated AA calculated AA Analyzed AA mg/g PE mg/gPE mg/g PE composition ^(B) composition ^(C) composition ^(A) Amino acidGMP GMP GMP mg/g PE GMP mg/g PE Total mg/g PE Total His  1.5 ± 0.1 222.9 ± 0.6 26.4 ± 0.6^(a)  21 ± 3^(a)  18 ± 3^(a) Ile  102 ± 2 0 5.6 ±0.5  108 ± 2^(a)  83 ± 7^(a,b)  70 ± 10^(b) Leu   23 ± 0.2 69  10 ± 1 102 ± 1^(a)  81 ± 6^(b)  77 ± 2^(b) Lys 60.1 ± 0.8 0   6 ± 1   66 ±1^(a)  51 ± 5^(a,b)  47 ± 8^(b) Met + Cys 20.5 ± 0.5 11   4 ± 1   36 ±1^(a)  28 ± 2^(b)  22 ± 3^(b) Phe + Tyr  2.6 ± 0.6 66 8.7 ± 0.7 70.6 ±0.9^(a)  63 ± 4^(b)  45 ± 3^(c) Thr  161 ± 2 0 4.1 ± 0.7  165 ± 2^(a)130 ± 10^(a) 120 ± 30^(a) Trp   0 ± 0 9 1.7 ± 0.1 10.7 ± 0.1^(a)  8.7 ±0.6^(a)  10 ± 2^(a) Val   85 ± 2 0   7 ± 1   91 ± 3^(a)  70 ± 6^(a,b) 70 ± 10^(b) ^(A) Composition obtained by analysis. Values are mean ±SD. Sample size was n = 2. ^(B) Calculated without including added aminoacids in the denominator (Method of Table 2, column F). ^(C) Calculatedincluding added amino acids in the denominator (Method of Table 2,column H). Same letter between the columns D, E, and F indicates that nodifference was detected between means (P > 0.05). A statisticallysignificant difference was detected between columns E and F for Tyr +Phe at α <0.05 but not at α <0.01.

For comparison, the amino acid composition of the GMP strawberry puddingwas calculated to include the changes in the denominator (Table 20,column E). Ignoring the contribution to the denominator resulted in a30% overestimation, on average, of the calculated amino acid compositioncompared to the observed values (Table 20, column D compared with F). Byincluding the contribution to the denominator, the corrected calculatedamino acid composition was not statistically different from the observedcomposition (P>0.05), except for Tyr (P<0.05) (Table 20, column Ecompared with F). The observed amino acid composition of the GMPstrawberry pudding (column F) met or exceeded all of the DRI targetvalues (Table 15).

The amino acid composition of GMP strawberry pudding was compared to anamino acid formula (see FIG. 10). The amino acid formula containedsignificantly more His, Leu, Met+Cys, Tyr, and Trp than the GMPstrawberry pudding (P<0.05). However, both amino acid formula and GMPstrawberry pudding met or exceeded DRI targets for all indispensableamino acids (P>0.05, Table 15).

Discussion

Manufacture of Purified GMP.

The low overall recovery of GMP (52±4%) was attributed to interactionsbetween GMP and the cation exchange column and resulted in a portion ofGMP binding to the column. The binding of GMP to the column wasattributed to heterogeneity of GMP. Some GMP molecules were lessnegatively charged than others at the operating pH of 4 and thereforebound to the cation exchange column. Operating at a higher pH may haveminimized GMP binding to the column by increasing the negative charge onGMP. This would also cause a decrease in electrostatic attractionbetween residual whey proteins and the cation exchange column as thepositive charge on these proteins would be decreased. Increasing theoperating pH to greater than 4 would have compromised purity. Purity wasa priority over recovery in the production of GMP used in the clinicaltrial.

UF/DF removed low molecular weight solutes and concentrated the GMPprior to the final drying step. UF/DF cannot remove contaminating wheyproteins, such as ALA and BLG, because these proteins are too large topermeate a 3 kDa membrane. On the other hand, low molecular weightsolutes, such as whey peptides, are small enough to be removed by UF/DFand may contain Phe.

Lyophilization produced a fine white powder with no flavor or odor anddissolved clearly in water (data not shown). However, the disadvantageof this drying method was the long processing time. Each other step inthe process could be completed in 1 d, but lyophilization took severaldays to complete. Despite the intensive time requirement, lyophilizationwas the most practical choice to dry purified GMP for use in this study.Spray drying was not used because of the potential losses of GMP andlimitations of the method when drying small quantities of product. Inlarge-scale manufacture, spray drying would be the method of choice.

Amino Acid Supplementation of Manufactured GMP.

The calculation method used for supplementation of GMP ignored the gramsof PE from added amino acids in the denominator, but was easilyimplemented and resulted in a GMP strawberry pudding that met orexceeded the DRI targets for all indispensable amino acids (Table 20).For GMP alone, ignoring the change to the denominator from added aminoacids did not result in a statistically significantly different GMPcomposition (Table 16, column F compared with H).

Ignoring the change in the denominator from addition of both nondairycreamer and supplemented amino acids resulted in a 30% overestimation ofamino acids compared to the observed value and 6 of the 9 amino acidswere statistically significantly below the observed value (Table 20,column D compared with F). On the other hand, when the PE contributed byadded amino acids were accounted for in the denominator, the correctedcalculated values matched the observed values (P>0.05) except for Tyr(P<0.05) (Table 20, column E compared with F). However, Tyr was notstatistically significantly different than the observed value at α<0.01(P>0.01). The lower than expected Tyr value was attributed to low purityof the Tyr supplement. Photodegradation of Tyr can occur and could havetaken place at some point during the manufacture or storage, which wouldlead to a lower than expected purity. Although the DRI for Tyr was metin the GMP food, increased Tyr supplementation would provide a higherlevel of Tyr.

Simplifications were reasonable for the supplementation calculations forGMP alone, due to the negligible impact on amino acid composition andease of implementation, but were not reasonable to make when performingthe mass balance calculations on the GMP food. GMP plus supplementedamino acids made up 15% (w/w) of the GMP pudding, whereas the nondairycreamer made up nearly 40% (w/w) of the pudding, and had a significantimpact on the final composition.

Although the nutritional requirement for amino acids is not a staticsubject, the mass balance calculation method of the present study isgenerically useful to supplement GMP foods to meet or exceed thenutritional needs of the human diet based on the most recent science.

Example 6: Acceptability of GMP Food of the Invention Compared toAA-Based Formula

In this Example, the inventors conducted sensory studies to compare theacceptability of Bettermilk™, a medical food of the present inventionmade by Cambrooke Foods, LLC, Ayer, Mass., to Phenex-2™, a conventionalamino acid formula for PKU diets, produced by Abbott Nutrition,Columbus, Ohio. Bettermilk contains GMP and supplemental amounts of theamino acids arginine, leucine, tyrosine, tryptophan, and histidine. Bothformulas were supplemented with vitamins and minerals to providenutritionally complete medical foods. The results indicate that GMPBettermilk is significantly more acceptable in both adult PKU andnon-PKU subjects compared with AA-based Phenex-2.

In the study, 27 non PKU adults and 4 PKU adults sampled both Bettermilkand Phenex-2. Participants then rated the acceptability of each producton a scale of 1-8. 1—dsilike extremely; 2—dislike a lot; 3—dislike;4—dislike a little; 5—like a little; 6—like; 7—like a lot; and 8—likeextremely. The results are shown in FIG. 11. As can be seen from thedata presented, participants rated the GMP food much higher than the AAformula on all acceptability criteria (odor, taste, after taste, andoverall), and the difference in acceptability was substantially greaterfor the PKU participants as compared to the non PKU participants. Thisprovides further evidence of the advantages of the GMP medical foods ofthe invention as compared to conventional amino acid formulas.

Example 7: Effects of GMP Foods as Compared with Amino Acids on GhrelinLevels in Individuals with PKU

Given the potential of GMP medical foods to promote satiety, theobjective of this study was to assess satiety using a visual analogscale (VAS) and compare plasma concentrations of ghrelin in individualswith PKU fed a breakfast of GMP medical foods as compared to an AA-basedbreakfast.

Using additional data from the study reported above in Example 4 above,this study demonstrates the ability of a breakfast of GMP foods topromote satiety and affect plasma concentrations of the appetitestimulating hormone ghrelin in those with PKU when compared to anAA-based breakfast. The eleven PKU subjects (8 adults and 3 boys ages11-14) profiled in Example 4 served as their own controls in aninpatient metabolic study with two 4-day treatments: an AA-based dietfollowed by a diet replacing all AA formula with GMP foods. Plasmaconcentration of ghrelin was obtained before and 180 min afterbreakfast. Satiety was assessed using a visual analog scale before,immediately after and 150 min after breakfast. Postprandial ghrelinconcentration was significantly lower (p=0.03) with GMP compared to anAA-based breakfast, with no difference in fasting ghrelin. Lowerpostprandial ghrelin concentrations were associated with greaterfeelings of fullness after breakfast suggesting greater satiety with GMPcompared to AAs. These results show sustained ghrelin suppression, andsuggest greater satiety with ingestion of a meal containing GMP comparedwith AAs.

Materials and Methods

Plasma Ghrelin Measurements

All postprandial blood samples were drawn 180 min after the start (150min after completion) of breakfast. For the last six subjects (Subject6-11) fasting blood samples were also obtained before breakfast on thelast 2 days of the AA diet (days 3 and 4) and on the last 2 days of theGMP diet (days 7 and 8). Total plasma ghrelin was measured in fasting(n=6) and postprandial (n=11) samples by radioimmunoassay (LincoResearch, St. Charles, Mo.); for each subject equal volumes of plasmawere combined for days 3+4 (AA diet) and days 7+8 (GMP diet) assupported by observed stability of the plasma AA profile on these days.Total ghrelin for subject 2 was removed from analysis because theinterpolated value was a clear statistical outlier that greatly exceededthe highest concentration on the standard curve.

Motivation-to-Eat VAS Questionnaires

Each subject completed a four-question motivation-to-eat VASquestionnaire three times: before breakfast, immediately followingbreakfast, and 2 h after finishing breakfast to assess subjectivemeasures of appetite and satiety. Each question consisted of a 100 mmline with opposing statements on either end. Subjects were asked toindicate with a vertical mark where on the line best described theirfeelings at the time with regards to the following questions: (1) Howstrong is your desire to eat?, (2) How hungry do you feel?, (3) How fulldo you feel? and (4) How much food do you think you can eat?(prospective food consumption, PFC). An appetite score to reflect thefour questions on the motivationto-eat VAS questionnaire was calculatedfor each questionnaire using the formula: Appetite score (mm)=[desire toeat+hunger+(100-fullness)+PFC]/4.

Statistical Analysis

All statistical analysis was conducted with the statistical program Rfor Mac OS X version 2.9 (R Project for Statistical Computing,Wirtschaftsuniversität, Vienna, Austria). Primary analyses wereperformed using two-tailed paired t-tests, pairing on subject. Testswere considered significant at p<0.05; values are means±SEM. Fasting andpostprandial plasma ghrelin values were compared within each diettreatment (e.g., fasting AA vs. postprandial AA) and between the diettreatments (e.g., fasting AA vs. fasting GMP) using a paired t-test,pairing on subject. Motivation-to-eat VAS questionnaires on the last dayof the AA diet and the last day of the GMP diet were compared withindiets (e.g., fasting AA vs. postprandial AA) and between diets (e.g.,fasting AA vs. fasting GMP). Secondary analysis used a linear mixedeffects model to examine the effect of: BMI, diet treatment, age,macronutrient intake at breakfast, plasma phe and plasma values(ghrelin, insulin and/or total AAs) on answers to daily VASquestionnaires and gherlin plasma value, controlling for a randomsubject effect. If there was no subject effect, a fixed effects linearmodel was used. The best model was found using backward elimination,eliminating insignificant variables.

Results

Motivation-to-Eat VAS Profiles

The motivation-to-eat VAS profiles were not significantly different atany time point between the AA diet (Day 4) and the GMP diet (Day 8).However as expected, the appetite profile significantly changed before,immediately after and 2 h after consuming either the AA or GMP breakfast(data not shown). Protein intake was identified as the most commonsignificant variable for the VAS questionnaires using the mixed effectsstatistical model. The protein content of breakfast showed a significantnegative association with the appetite score immediately after breakfast(p=0.01) such that the appetite score decreased with greater proteinintake at breakfast. In the final model for the appetite scoreimmediately after breakfast, other significant factors in addition toprotein content included BMI, age and the interaction between diettreatment and the day of the study. BMI significantly affected VASanswers at all time points in the mixed effects model analysis. A higherBMI was associated with a greater desire to eat, hunger, and appetitescore and lower feelings of fullness.

Plasma Ghrelin

The concentration of ghrelin in plasma obtained after an overnight fastwas not significantly different between the AA and GMP diets (see FIG.12) and there were no significant associations between fasting plasmaghrelin concentrations and a variety of variables. In particular, therewas no direct association found between fasting ghrelin and BMI in thisdiverse sample population. The total concentration of AAs in fastingplasma was also not significantly different between the two diets (datanot shown). Postprandial plasma ghrelin concentration, taken 180 minfollowing the start of the AA breakfast, was not different from fastingghrelin prior to eating the AA breakfast (FIG. 12). In contrast, the GMPbreakfast induced significantly lower postprandial plasma ghrelinconcentration, an expected response following a meal. In addition,postprandial ghrelin following the GMP breakfast was significantly lowerthan postprandial ghrelin following the AA breakfast. There were nosignificant associations between post-prandial ghrelin concentrationsand BMI. Using backward elimination with a linear mixed effects model,the only factor predicting postprandial ghrelin was diet treatment, inthat postprandial plasma ghrelin was lower with the GMP breakfast.Postprandial plasma ghrelin concentration was a significant factor inthe prediction of fullness 2 h after breakfast (see FIG. 13). Higherfullness scores were significantly associated with lower postprandialghrelin concentrations, diet treatment, and the interaction betweenghrelin and diet.

Discussion

The absence of a protein source in a meal may result in increasedfeelings of hunger throughout the day. The improved taste and preferencecombined with the ability to make a variety of good tasting foods withGMP supports the notion that GMP may improve the dietary management ofPKU by providing a protein source that can be more easily spacedthroughout the day. Moreover, we report for the first time thatingestion of intact protein from GMP compared with synthetic AAs resultsin sustained suppression of ghrelin following a meal in those with PKU.

Ghrelin is the only known orexigenic hormone, with highestconcentrations in the fasted state and in anticipation of a meal,whereas concentrations are suppressed following a meal. We found nodifference in fasting ghrelin concentration when comparing the AA andGMP breakfasts in 3 children (ages 11-14) and 8 adults with PKU whoserved as their own control.

The data provide novel information about the response of ghrelin to ameal. We demonstrate that isocaloric breakfast treatments containing theintact protein GMP compared with synthetic AAs induce a differentghrelin response. Ghrelin levels decrease after a meal in proportion tocaloric content and protein and carbohydrate suppress ghrelin to asimilar extent in studies with isocaloric substitution of 20% energy orgreater from protein or carbohydrate. Thus, the higher proportion ofenergy provided by carbohydrate in the GMP breakfast (7.8%) is unlikelyto account for the differential ghrelin response observed in this study.

Ghrelin concentration was only measured at two time points, fasting and180 min after the start of breakfast, therefore the nadir was likelymissed. However, the observed significant decline in ghrelinconcentrations between these two time points with the GMP breakfast, butnot the AA, shows that the intake of an AA breakfast does not allowsustained suppression of ghrelin 180 min after breakfast. In fact, theghrelin hunger signal 3 h after the AA meal was no different than aftera 12 h fast. Greater ghrelin suppression following a meal with intactprotein compared to AAs may be due to variations in the rate ofabsorption of synthetic AAs compared with GMP. The appearance of AAs inplasma occurs within 1 h following consumption of AAs and approximately2 h with consumption of a comparable intact protein. Fast absorption ofAAs has been shown to negatively affect protein retention andutilization in rats and humans.

Although the most significant consequence of long-term AA consumptionmay be reduced protein retention, the sharp rise of plasma AAsconcentration also affects physiological signals of satiety. Theaminostatic hypothesis proposes that an increase in plasma AAconcentrations is accompanied by a greater stimulation of GI hormonesand decreased appetite followed by a return of appetite when plasma AAconcentrations fall (S. M. Mellinkoff, M. Frankland, D. Boyle, M.Greipel, Relationship between serum amino acid concentration andfluctuations in appetite. 1956, Obes. Res. 5 (1997) 381-384). Thus, wheyproteins, such as GMP, may decrease appetite due to rapid absorption andsustained levels of plasma AAs, whereas synthetic AAs cause an acuterise in plasma AAs which disappear from plasma faster and to a greaterextent compared to intact protein, resulting in increased appetiteshortly after a meal. Supportive of this hypothesis, a breakfastcontaining GMP induced higher total postprandial plasma AA and lowerghrelin concentrations compared with synthetic AAs. Moreover, our datashow an association between lower postprandial ghrelin concentration andgreater feelings of fullness suggesting that a GMP meal sustains satietywhen compared with AAs.

Ghrelin suppression is regulated by postgastric feedback (D. L.Williams, D. E. Cummings, H. J. Grill, J. M. Kaplan, Meal-relatedghrelin suppression requires postgastric feedback (Endocrinology 144(2003) 2765-2767), requiring luminal nutrients in the distal intestine,not in the stomach or duodenum. The rapid rise of plasma AAs followingconsumption of an AA-based formula suggests that luminal nutrients arepresent for a shorter time, therefore limiting their ability to suppressghrelin.

In addition, ghrelin works in a reciprocal manner with insulin (D. E.Cummings, J. Q. Purnell, R. S. Frayo, K. Schmidova, B. E. Wisse, D. S.Weigle, A preprandial rise in plasma ghrelin levels suggests a role inmeal initiation in humans, Diabetes 50 (2001) 1714-1719). Similarly, ourresults show that postprandial plasma insulin concentration is higherand ghrelin lower after a breakfast containing GMP compared with anAA-based breakfast. Thus, GMP foods may improve insulin and ghrelinregulation, satiety signaling, and protein retention in individuals withPKU relative to a conventional amino acid diets.

CONCLUSION

The nutritional management of PKU is in need of new dietary optionsbesides synthetic AAs to facilitate ingestion of a low-phe source ofprotein throughout the day in order to improve metabolic control andcontrol hunger. These results confirm the importance of proteinconsumption in a meal to improve satiety, and provide novel evidencethat a GMP breakfast suppresses plasma levels of the satiety hormoneghrelin for a longer period of time compared with an AA breakfast. Foodproducts made with the intact, low-phe protein GMP are a first step toproviding a more physiologically complete diet that improves dietaryoptions, and facilitates protein distribution and metabolic control ofPKU.

Example 8: Recommended Amino Acid Supplementation for the PresentInvention

In this prophetic example, the inventors present recommended amino acidsupplementation amounts for the present invention. In particular, theinventors provide recommended variations from the supplementationamounts used in the previous working examples.

Methionine.

The inventors do not recommend that methionine be added to the medicalfoods of the present invention. It has recently been determined that theminimum requirement for methionine plus cysteine for school-age childrenand adults is significantly lower than previously thought (Turner, etal., Am J Clin Nutr 2006; 83:619-23; Ball, et al., J Nutr 2006; 136(suppl 2):1682S-93S). This suggests that GMP contains an adequate amountof methionine, and that methionine supplementation is not required. Asmethionine is a sulfur-containing amino acid with an unpleasant taste,not supplementing the GMP-based foods with methionine will furtherincrease the palatability of the foods.

Arginine.

In contrast to the previously presented examples, the inventorsrecommend supplementing the GMP in the medical foods of the inventionwith additional arginine. Specifically, the inventors propose addingarginine to the medical foods of the invention so that the total weightratio within the food of the arginine to the protein is preferably fromabout 60 to 90 milligrams arginine/gram total protein, and morepreferably about 75 milligrams arginine/gram total protein.

Although arginine is recognized in the art as a nutritionallydispensable amino acid (Tharakan et al., Clin Nutr 2008; 27:513-22),arginine has multiple non-nutritional functions, which include servingas a substrate for synthesis of protein, urea, and nitric oxide (thecofactor for PAH, tetrahydrobiopterin, is also the cofactor for nitricoxide synthetase). Arginine is synthesized in the kidney from intestinalcitrulline derived from glutamine and oxidized to ornithine in the ureacycle. Consistent with minimal arginine in GMP, plasma concentrations ofarginine and ornithine reported in the clinical trials of Example 4 weresignificantly lower with ingestion of the GMP compared with the AA diet(see Table 14). Thus, the inventors conclude that GMP should besupplemented with arginine for utilization in the PKU diet.

Leucine.

The inventors recommend that the medical foods of the present inventionbe supplemented with an amount of leucine that is substantially morethan that indicated by any published recommended intake requirement orthan amount used in any of the above working examples. Specifically, theinventors propose adding leucine to the medical foods of the inventionso that the total weight ratio within the food of leucine to the proteinis preferably from about 100 to 200 milligrams leucine/gram totalprotein, and more preferably about 100 milligrams leucine/gram totalprotein.

The inventors have determined that elevated plasma levels of leucine maycompetitively inhibit Phe transport across the intestinal mucosa andblood brain barrier via a certain carrier protein. Thus, leucinesupplementation beyond levels of nutritional need may surprisinglyreduce Phe levels in both plasma and brain, the primary organ where Pheexerts its neurotoxic effects. The higher recommended leucine levels,then, may lead to decreased levels of Phe in plasma and brain inindividuals with PKU using the GMP-based foods of the invention.

Furthermore, recent evidence demonstrates that leucine stimulatessynthesis of skeletal muscle protein by enhancement of mRNA translationinitiation rates (Norton L E et al., J Nutr 2009; 139:1103-1109 andCrozier, S J et al., J Nutr 2005; 135:376-382). Improved synthesis ofskeletal muscle protein in those with PKU would lower blood phe levelsand may increase lean body mass.

Tyrosine.

A supplemented amount of tyrosine is not added to medical foods for thetreatment of tyrosine metabolism disorders, such as tyrosinemia. Forother applications, including for a PKU diet, the inventors recommendthat the medical foods of the present invention be supplemented with anamount of tyrosine that is somewhat more than the amount used in any ofthe above working examples. Specifically, the inventors propose addingtyrosine to non heat-treated medical foods of the invention so that theinitial total weight ratio within the food of tyrosine to the protein ispreferably from about 62 to 93 milligrams tyrosine/gram total protein,and more preferably about 85 milligrams tyrosine/gram total protein.

Tyrosine is an important AA in the PKU diet because it is indispensableand a precursor of adrenaline, norepinephrine, melanin, and thyroxine.In Example 4 above, the inventors found that concentrations of tyrosinein plasma obtained in the postprandial or fasting samples were notsignificantly different with ingestion of the GMP or AA diets (see Table14 above). Concentrations of tyrosine in plasma after an overnight fastwere decreased compared with postprandial concentrations with ingestionof both the GMP and the AA diet. However, the GMP diet resulted in amean fasting tyrosine concentration that was below the normal range.Thus, the inventors recommend tyrosine supplementation above the 150%DRI supplementation level of the foods tested in Example 4.

In addition, in Example 5 above, the inventors found that when the PEcontributed by added amino acids were accounted for in the denominator,the corrected calculated values matched the observed values (P>0.05) ofall the measured amino acids, except for Tyr (P<0.05) (see Table 20above, column E compared with F). The lower than expected Tyr valuesuggests that Tyr degraded at some point during the manufacture orstorage process. Although the DRI for Tyr was met in the GMP food (whichwere supplemented at 150% of DRI), increased Tyr supplementation wouldprovide a higher level of Tyr.

Tryptophan.

A supplemented amount of tryptophan is not added to medical foods forthe treatment of tryptophan metabolism disorders, such ashypertryptophanemia. For other applications, including for a PKU diet,the inventors propose optionally adding tryptophan to the medical foodsof the invention so that the initial total weight ratio within the foodof tryptophan to the protein is preferably from about 12 to 14milligrams tryptophan/gram total protein, and more preferably about 12milligrams tryptophan/gram total protein.

In Example 4, the inventors observed a 29% decrease in postprandialplasma trp levels with the GMP compared with AA diet. The trp levels inthe GMP diet was well below the range of trp in AA formulas (˜15 mgtrp/g protein). There is also evidence that tip is important insynthesis of the neurotransmitter, serotonin (Passcuchi et al., Intl JNeuropsychopharmacology (2009), 12:1067-79). Thus, the recommendedlevels are significantly above both the range established using 130-160%of the recommended minimum intake guidelines published by WHO (WorldHealth Organization, Protein and Amino Acid Requirements in HumanNutrition, Geneva, Switzerland: United Nations University, 2007) or therecommended amount based on 130% of the 2002 DRI (Institute of Medicine,Dietary Reference Intakes for Energy, Carbohydrates, Fiber, Fat, Proteinand Amino Acids, Washington, D.C.: National Academy Press, 2002).

Histidine.

A supplemented amount of histidine is not added to medical foods for thetreatment of histidine metabolism disorders, such as histidinemia. Forother applications, including for a PKU diet, the inventors proposeoptionally adding histidine to the medical foods of the invention sothat the initial total weight ratio within the food of histidine to theprotein is preferably from about 20 to 24 milligrams histidine/gramtotal protein, and more preferably about 23 milligrams histidine/gramtotal protein. The preferred range is based on 130-160% of therecommended minimum intake guidelines published by WHO (World HealthOrganization, Protein and Amino Acid Requirements in Human Nutrition,Geneva, Switzerland: United Nations University, 2007). The morepreferred value is based on 130% of the 2002 DRI (Institute of Medicine,Dietary Reference Intakes for Energy, Carbohydrates, Fiber, Fat, Proteinand Amino Acids, Washington, D.C.: National Academy Press, 2002). Unlikethe recommendations for the other amino acids, these recommended valuesfor histidine do not vary from what is contained in the GMP medicalfoods used in the working examples.

Based on the amino acid supplementation amounts presented in thisExample, improved GMP-based medical foods can be produced to provide theneeded protein for individuals with PKU while functioning to minimizePhe levels in the blood plasma and brain tissue.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific materials and methods described herein. Such equivalents areconsidered to be within the scope of this invention and encompassed bythe following claims.

We claim:
 1. A medical food for the management of a metabolic disorder,comprising glycomacropeptide (GMP) and two or more supplemental aminoacids, wherein: (a) the total weight of the supplemental amino acids isfrom about 22% to 38% of the total weight of the protein from the GMPand the supplemental amino acids together; and (b) one of thesupplemental amino acids is arginine and the weight ratio within themedical food of the amino acid arginine to total protein from the GMPand the supplemental amino acids together is from about 60 to 90milligrams arginine/gram total protein.
 2. The medical food of claim 1,wherein the medical food does not contain a supplemented amount of theamino acid tyrosine.
 3. The medical food of claim 1, wherein the medicalfood is in the form of a beverage, a bar, a wafer, a pudding, a gelatin,a cracker, a fruit leather, a nut butter, a sauce, a salad dressing, acrisp cereal piece, a flake, a puff, a pellet, or an extruded solid. 4.The medical food of claim 1, wherein the two or more supplemental aminoacids comprise two or more amino acids selected from the groupconsisting of arginine, leucine, tyrosine, tryptophan, and histidine. 5.The medical food of claim 4, wherein the two or more supplemental aminoacids comprise arginine and leucine.
 6. The medical food of claim 4,wherein the two or more supplemental amino acids comprise tyrosine.
 7. Amethod of making a medical food for the management of a metabolicdisorder, the method comprising the step of mixing glycomacropeptide(GMP) and two or more supplemental amino acids to make a food, wherein:(a) the total weight of the supplemental amino acids is from about 22%to 38% of the total weight of the protein from the GMP and thesupplemental amino acids together; and (b) one of the supplemental aminoacids is arginine and the weight ratio within the medical food of theamino acid arginine to total protein from the GMP and the supplementalamino acids together is from about 60 to 90 milligrams arginine/gramtotal protein.
 8. The method of claim 7, wherein the two or moresupplemental amino acids comprise two or more amino acids selected fromthe group consisting of arginine, leucine, tyrosine, tryptophan, andhistidine.
 9. The method of claim 7, wherein the two or moresupplemental amino acids comprise arginine and leucine.
 10. The methodof claim 7, wherein the two or more supplemental amino acids do notcomprise tyrosine.
 11. A method of treating a metabolic disorder,comprising administering to a human having a metabolic disorder amedical food comprising glycomacropeptide (GMP) and two or moresupplemental amino acids, wherein the metabolic disorder is selectedfrom a Phenylalanine metabolism disorder, a Tyrosine metabolismdisorder, a Tryptophan metabolism disorder, and a Histidine metabolismdisorder, and wherein: (a) the total weight of the supplemental aminoacids in the medical food is from about 22% to 38% of the total weightof the protein from the GMP and the supplemental amino acids together;and (b) one of the supplemental amino acids is arginine and the weightratio within the medical food of the amino acid arginine to totalprotein from the GMP and the supplemental amino acids together is fromabout 60 to 90 milligrams arginine/gram total protein.
 12. The method ofclaim 11, wherein the two or more supplemental amino acids comprisearginine and leucine.
 13. The method of claim 11, wherein the two ormore supplemental amino acids do not comprise tyrosine.
 14. The methodof claim 13, wherein the metabolic disorder is a Tyrosine metabolismdisorder.